EA048431B1 - CLADE F ADENO-ASSOCIATED VIRUS (AAV) VECTOR AND ITS APPLICATIONS - Google Patents

Abstract

A recombinant adeno-associated virus (rAAV)-based vector comprising an AAVhu68 capsid produced in a production system comprising the nucleotide sequence of SEQ ID NO: 1, or a sequence at least 75% identical thereto, which encodes SEQ ID NO: 2. The AAVhu68 capsid comprises subpopulations of highly deamidated asparagine residues in asparagine-glycine pairs in the amino acid sequence of SEQ ID NO: 2. Compositions comprising rAAV and their uses are also provided. Additionally, an rAAV is provided having an engineered AAV capsid comprising at least one subpopulation of vpl or vp2 proteins having Vai at amino acid position 157 relative to the vpl numbering of AAVhu68.

Description

Declaration of Federally Sponsored Research or Development

This application contains work supported by the Defense Advanced Research Projects Agency (DARPA) under W911NF-13-2-0036. The U.S. Government has certain rights in this invention.

State of the art

Adeno-associated virus (AAV), a member of the parvovirus family, is a small, icosahedral, capsid-less virus with a single-stranded linear DNA (ssDNA) genome of approximately 4.7 kilobase pairs (kbp). The wild-type genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. Rep consists of four overlapping genes encoding the rep proteins required for the AAV life cycle, and cap contains overlapping nucleotide sequences of the capsid proteins: VP1, VP2, and VP3, which self-assemble to form a capsid of icosahedral symmetry.

AAV is classified as a member of the genus Dependovirus because the virus was detected as a contaminant in purified adenovirus cultures. The AAV life cycle includes a latent phase in which the AAV genome, following infection, is site-specifically integrated into the host chromosomes, and an infectious phase in which, following infection with either adenovirus or herpes simplex virus, the integrated genomes are subsequently retrieved, replicated, and packaged into infectious viruses. The properties of non-pathogenicity, infectivity for a broad range of hosts, including non-dividing cells, and potential for site-specific chromosomal integration make AAV an attractive tool for gene transfer.

Recombinant adeno-associated virus (rAAV) vectors produced by replication defective human parvovirus have been described as suitable gene delivery vehicles. Typically, the functional rep genes and cap gene are deleted from the vector, resulting in a replication-incompetent vector. These features are provided during the vector production system but are absent from the final vector.

To date, there have been several different well-characterized AAVs isolated from humans or non-human primates (NHPs). AAVs of different serotypes have been found to exhibit different transfection efficiencies and to have tropisms for different cells or tissues. Many different clades of AAV have been described in WO 2005/033321, including clade F, which is identified therein as having only three members: AAV9, AAVhu31, and AAVhu32. A structural analysis of AAV9 is presented in M. A. DiMattia et al., J. Virol. (June 2012) vol. 86 no. 12 6947-6958. This paper reports that AAV9 has 60 copies (in total) of three different proteins (vp) that are encoded by the cap gene and have overlapping sequences. They include VP1 (87 kDa), VP2 (73 kDa), and VP3 (62 kDa), which are present in a predicted ratio of 1:1:10, respectively. The entire sequence of VP3 is contained within VP2, and the entire sequence of VP2 is contained within VP1. VP1 has a unique N-terminal domain. The refined coordinates and structure factors are available under RCSB PDB accession number 3UX1.

Several different AAV9 variants have been engineered to be non-targeted or to target different tissues. See, for example, N. Pulicheria, Engineering Liver-detargeted AAV9 Vectors for Cardiac and Musculoskeletal Gene Transfer, Molecular Therapy, Vol, 19, no. 6, p. 1070-1078 (June 2011). Engineering of AAV9 variants for gene delivery across the blood-brain barrier has also been reported. See, for example, B.E. Deverman et al, Nature Biotech, Vol. 34, No. 2, p 204-211 (published February 1, 2016) and Caltech press release, A. Wetherston, www.neurologycentral.com/2016/02/10/successful-delivery-of-genes-through-the-blood-brain-barrier/, accessed May 10, 2016. See also WO 2016/0492301 and US 8734809.

It is desirable to develop AAV-based constructs for the delivery of heterologous molecules.

The essence of the invention

A novel AAVhu68 capsid and rep sequences are described that may be useful for a manufacturing process and in vectors for delivering nucleic acid molecules to host cells. In some embodiments, a recombinant AAV is provided that has an AAVhu68 capsid that is encoded by the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 1, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the invention provides a recombinant adeno-associated virus (rAAV) that comprises: (A) an AAV68 capsid comprising one or more of: (1) AAV hu68 capsid proteins comprising: AAVhu68 vp1 proteins obtained by expression from a nucleic acid sequence that encodes the predicted amino acid sequence from 1 to 736 of SEQ ID NO: 2, vp1 proteins obtained from SEQ ID NO: 1, or vp1 proteins obtained from a nucleic acid sequence at least 70% identical to SEQ ID NO: 1 that encodes the predicted amino acid sequence from 1 to 736 of SEQ ID NO: 2, AAVhu68 vp2 proteins,

- 1 048431 obtained by expression from a nucleic acid sequence that encodes a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 2, vp2 proteins obtained from a sequence comprising at least nucleotides from 412 to 2211 of SEQ ID NO: 1, or vp2 proteins obtained from a nucleic acid sequence at least 70% identical to at least nucleotides from 412 to 2211 of SEQ ID NO: 1 that encodes a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 2, AAVhu68 vp3 proteins obtained by expression from a nucleic acid sequence that encodes a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO: 2, vp3 proteins obtained from a sequence comprising at least at least nucleotides 607 to 2211 of SEQ ID NO: 1, or vp3 proteins derived from a nucleic acid sequence at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO: 1 that encode a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO: 2; and/or (2) AAV capsid proteins comprising a heterogeneous population of vp1 proteins optionally comprising valine at position 157 and/or glutamic acid at position 67, a heterogeneous population of vp2 proteins optionally comprising valine at position 157, and a heterogeneous population of vp3 proteins, wherein at least a subpopulation of the vp1 and vp2 proteins comprises valine at position 157 and optionally further comprises glutamic acid at position 67 based on the vp1 capsid numbering of SEQ ID NO: 2; and/or (3) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 2, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO: 2, wherein the vp1, vp2, and vp3 proteins comprise subpopulations with amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairs of SEQ ID NO: 2, and optionally further include subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid substitution; and (B) a vector genome in an AAVhu68 capsid, wherein the vector genome comprises a nucleic acid molecule comprising AAV inverted terminal repeat sequences and a non-AAV nucleic acid sequence encoding a product operably linked to sequences that direct expression of that product in a host cell. For example, four residues (N57, N329, N452, N512) typically show high levels of deamidation. Additional residues (N94, N253, N270, N304, N409, N477, and Q599) also show levels of deamidation of up to ~20% across different lots.

In some embodiments, the deamidated asparagines are deamidated to aspartic acid, isoaspartic acid, an aspartic acid/isoaspartic acid interconvertible pair, or combinations thereof. In some embodiments, the deamidated glutamine(s) are deamidated to (a)-glutamic acid, γ-glutamic acid, an (a)-glutamic acid/γ-glutamic acid interconvertible pair, or combinations thereof.

In certain embodiments, the AAVhu68 capsid comprises subpopulations having one or more of: (a) at least 65% of the asparagines (N) in the asparagine-glycine pairs located at position 57 of the vp1 proteins are deamidated, as determined by the numbering of SEQ ID NO: 2; (b) at least 75% of the N in the asparagine-glycine pairs at position 329 of the vp1, v2, and vp3 proteins are deamidated, as determined by the residue numbering of the amino acid sequence of SEQ ID NO: 2; (c) at least 50% of the N in the asparagine-glycine pairs at position 452 of the vp1, v2, and vp3 proteins are deamidated, as determined by the residue numbering of the amino acid sequence of SEQ ID NO: 2; and/or (d) at least 75% of the N in the asparagine-glycine pairs at position 512 of the vp1, v2, and vp3 proteins are deamidated, as determined by the residue numbering of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the hu68 capsid comprises a vp1 subpopulation in which 75% to 100% of the N at position 57 of the vp1 proteins is deamidated, as determined by mass spectrometry. In some embodiments, the hu68 capsid comprises a subpopulation of vpl proteins, vp2 proteins, and/or vp3 proteins in which 75% to 100% of the N at position 329, based on the residue numbering of SEQ ID NO: 2, is deamidated, as determined by mass spectrometry. In some embodiments, the hu68 capsid comprises a subset of vp1 proteins, vp2 proteins, and/or vp3 proteins in which between 75% and 100% of the N at position 452, based on the numbering of SEQ ID NO: 2, is deamidated, as determined using mass spectrometry. In some embodiments, the hu68 capsid comprises a subset of vp1 proteins, vp2 proteins, and/or vp3 proteins in which between 75% and 100% of the N at position 512, based on the numbering of SEQ ID NO: 2, is deamidated. In some embodiments, the nucleic acid sequence encoding

- 2 048431 proteins, is SEQ ID NO: 1, or a sequence of at least 80% to at least 99% identical to the sequence of SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the sequence is at least 80% to 97% identical to SEQ ID NO: 1. In certain embodiments, the rAAVhu68 capsid further comprises at least a subpopulation of vp1, vp2 and/or vp3 proteins having amino acid modifications of SEQ ID NO: 2 comprising at least about 50% to 100% deamidation at at least four positions selected from one or more of N57, 329, 452, 512, or combinations thereof. In certain embodiments, the hu68 capsid comprises subpopulations of the vp1, vp2, and/or vp3 proteins that further comprise from 1% to about 40% deamidation at at least one or more positions N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N336, N409, N410, N477, N515, N598, Q599, N628, N651, N663, N709, or combinations thereof. In certain embodiments, the hu68 capsid comprises subsets of vp1, vp2, and/or vp3 proteins that further comprise one or more modifications selected from one or more modifications in one or more of the following: acetylated lysine, phosphorylated serine and/or threonine, isomerized aspartic acid, oxidized tryptophan and/or methionine, or an amidated amino acid. In certain embodiments, rAAVhu68 comprises about 60 total capsid proteins in a ratio of about 1 vp1 to about 1 to 1.5 vp2 to 3 to 10 vp3 proteins. In certain embodiments, the AAVhu68 capsid comprises about 60 total capsid proteins in a ratio of about 1 vp1 to about 1 vp2 to 3 to 9 vp3 proteins. In certain embodiments of the invention, the vector genome comprises AAV ITR sequences from an AAV source other than AAVhu68.

In certain embodiments, the invention provides a composition that comprises a mixed population of recombinant adeno-associated virus hu68 (rAAVhu68), wherein each rAAVhu68 is independently selected from rAAVhu68 as described herein. In certain embodiments, an average AAVhu68 capsid comprises a total of about 60 capsid proteins in a ratio of about 1 vp1 to about 1 to 1.5 vp2 to 3 to 10 vp3 proteins. In certain embodiments, an average AAVhu68 capsid comprises a total of about 60 capsid proteins in a ratio of about 1 vp1 to about 1 vp2 to 3 to 6 vp3 proteins. In certain embodiments, the composition is formulated for intrathecal delivery and the vector genome comprises a nucleic acid sequence encoding a product for delivery to the central nervous system. In certain embodiments, the composition is formulated for intravenous delivery. In certain embodiments, the vector genome comprises a nucleic acid sequence encoding an anti-HER2 antibody. In certain embodiments, the composition is formulated for intranasal or intramuscular delivery. In certain embodiments, the composition comprises at least the parent rAAVhu68 vector and an optional carrier, excipient, and/or preservative.

Certain embodiments of the invention provide for the use of rAAVhu68 or a composition as described herein to deliver a desired gene product to a subject in need thereof.

In certain embodiments, a rAAV production system suitable for producing recombinant AAVhu68 is provided. The production system comprises: (a) an AAVhu68 capsid nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; (b) a nucleic acid molecule suitable for packaging into an AAVhu68 capsid, wherein the nucleic acid molecule comprises at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences that direct expression of the product in a host cell; and (c) sufficient AAV rep functions and ancillary functions to ensure packaging of the nucleic acid molecule into a recombinant AAVhu68 capsid. In certain embodiments, the nucleic acid sequence (a) comprises at least SEQ ID NO: 1, or a sequence that is at least 70% to at least 99% identical to the sequence of SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the system further comprises a nucleic acid sequence from about nucleotide 607 to about nucleotide 2211 of SEQ ID NO: 1, encoding AAVhu68 vp3 from about amino acid 203 to about amino acid 736 of SEQ ID NO: 2. In certain embodiments, the system comprises human embryonic kidney 293 cells or a baculovirus system.

In some embodiments, the invention provides a method for reducing deamidation of an AAVhu68 capsid. The method comprises producing an AAVhu68 capsid from a nucleic acid sequence comprising modified AAVhu68 vp codons, wherein the nucleic acid sequence comprises independently modified glycine codons in one to three arginine-glycine pairs located at positions 58, 330, 453, and/or 513 of SEQ ID NO: 2, such that the modified

- 3 048431 the modified codon encodes an amino acid other than glycine. In certain embodiments, the method comprises producing an AAVhu68 capsid from a nucleic acid sequence comprising modified vp AAVhu68 codons, wherein the nucleic acid sequence comprises independently modified arginine codons in one to three arginine-glycine pairs located at positions 57, 329, 452 and/or 512 of SEQ ID NO: 2 such that the modified codon encodes an amino acid other than arginine. In certain embodiments, each modified codon encodes a different amino acid. In certain embodiments, two or more modified codons encode the same amino acid. In certain embodiments, a mutant AAVhu68 capsid as described herein comprises a mutation in an arginine-glycine pair such that glycine is replaced with alanine or serine. The mutant AAVhu68 capsid may comprise one, two, or three mutants, wherein the reference AAVhu68 initially comprises four NG pairs. In certain embodiments, the mutant AAVhu68 capsid comprises only one mutation in an NG pair. In certain embodiments, the mutant AAV capsid comprises mutations in two different NG pairs. In some embodiments, the mutant AAVhu68 capsid comprises mutations in two different NG pairs that are located at a structurally separated location in the AAVhu68 capsid. In certain embodiments, the mutation is not in the VP1 unique region. In certain embodiments, one of the mutations is in the VP1 unique region. Optionally, the mutant AAVhu6 capsid does not comprise modifications in the NG pairs, but comprises mutations to minimize or eliminate deamidation at one or more asparagines or glutamines located outside of the NG pair.

In certain embodiments, the invention provides a mutant rAAVhu68 that includes a modified rAAVhu68 capsid with reduced deamidation compared to an unmodified AAVhu68 capsid that is produced using the method described herein.

In yet another further aspect, a method for increasing the yield and/or packaging efficiency of a recombinant adeno-associated virus (rAAV) vector is provided. The method comprises engineering an AAV capsid gene to express a vp1 protein with Val at amino acid position 157, wherein the amino acid residue numbering is based on the full-length vpl of AAVhu68 [SEQ ID NO: 2]. In certain embodiments, a clade F rAAV is provided having a glutamic acid (Glu or E) at amino acid position 67 based on the numbering of SEQ ID NO: 2.

In yet another further embodiment of the invention, there is provided an engineered rAAV produced in accordance with this method.

In a further embodiment of the invention, there is provided an AAVhu68 particle that expresses an anti-HER2 antibody, useful for the treatment and/or prevention of HER2+ cancers.

In yet another further embodiment of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding the AAVhu68 rep protein or a functional fragment thereof under the control of exogenous regulatory control sequences that direct their expression in a host cell. In one embodiment of the invention, the rep protein has the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof.

These and other aspects of the present invention will be apparent from the following detailed description of the invention.

Brief description of graphic materials

Figure 1 shows an alignment showing the amino acid sequence of the vp1 capsid protein of AAVhu68 [SEQ ID NO: 16] (labeled hu.68.vp1 in the alignment), with AAV9 [SEQ ID NO: 6], AAVhu31 (labeled hu.31 in the alignment) [SEQ ID NO: 10], AAVhu32 (labeled hu.32 in the alignment) [SEQ ID NO: 11]. Compared with AAV9, AAVhu31 and AAVhu32, two mutations (A67E and A157V) were found as critical in AAVhu68 and are circled in the figure.

Figure 2A-C shows the alignment of the nucleic acid sequence encoding the vp1 capsid protein from AAVhu68 with AAV9, AAVhu31 [SEQ ID NO: 12], and AAVhu32 [SEQ ID NO: 13].

Fig. 3A, B are graphs showing the yields of AAVhu.68 compared to AAV9. The experiment was performed as described in Example 2, n=6. The P value was calculated and shown in the figures.

Figure 3A shows the yields of AAVhu.68 and AAV9 from total lysate. The P value was calculated to be 0.4173 and was determined to be not significant.

Figure 3B shows the yields of AAVhu.68 and AAV9 from the culture supernatant. The yield of AAVhu.68 in the supernatant was significantly higher than that of AAV9 with a p value of 0.0003.

Figures 4A-C show immunohistochemical staining of various organs (heart, liver, lung, and muscle) in mice treated with 5x10 ¹¹ GC AAVhu68.CB7.nLacZ. Samples were prepared and processed as described in Example 3. Samples counterstained with eosin are shown in red.

- 4 048431 volume. Positive staining for LacZ, shown in blue, indicates successful transduction of AAVhu68.

Figure 4A shows immunohistochemical staining of various organs (heart, liver, lung, and muscle) from mice injected with 5x 10 ¹¹ GC AAVhu68.CB7.nLacZ intravenously (i.v.). All organs examined demonstrated transduction of AAVhu68 while a tropism favoring heart and liver over lung and muscle was observed.

Figure 4B shows immunohistochemical staining of various organs (heart, liver, lung, and muscle) of mice injected with 5x 10 ¹¹ GC AAVhu68.CB7.nLacZ intramuscularly (i.m.). Heart, liver, and muscle showed high levels of AAVhu68 transduction, while no visible transduction was detected in the lung.

Figure 4C shows immunohistochemical staining of various organs (heart, liver, lung, and muscle) in mice injected with 5x1011 GC AAVhu68.CB7.nLacZ intranasally (i/n). Diffuse transduction was observed in the heart, liver, muscle, and lung.

Figures 5A-C show fluorescence microscopy images of different brain regions (hippocampus, Figure 5A; motor cortex, Figure 5B; and cerebellum, Figure 5C) of mice injected with AAVhu68.GFP or AAV9.GFP at doses of ^1x1010 GC or ^1x1011 GC. Samples were prepared and processed as described in Example 4. A positive GFP signal, shown in green, indicates successful transduction of AAV vectors.

Figure 5A shows fluorescence microscopy images of hippocampal sections from mice injected with 1x10 ¹⁰ GC or 1x10 ¹¹ GC of AAVhu68.GFP or AAV9.GFP. Corresponding nucleic acid dye-stained samples from untreated mice, shown in blue, were provided as negative controls. Transduction of AAV vectors was observed in all samples tested except one of the mice injected with 1x10 ¹⁰ GC of AAV9.GFP.

Fig. 5B shows fluorescence microscopy images of the motor cortex of mice injected with AAVhu68.GFP or AAV9.GFP at doses of 1x10 ¹⁰ GC or 1x1011 GC. Better transduction of AAVhu68.GFP was observed compared to AAV9.

Figure 5C shows fluorescence microscopy images of cerebellar sections from mice injected with AAVhu68.GFP or AAV9.GFP at doses of 1x 10 ¹⁰ GC or 1x 10 ¹¹ GC. Successful transduction of AAVhu68.GFP was observed when mice were injected with 1x 10 ¹¹ GC vector.

Figures 6A-D show microscopic images of various organs (liver, kidney, heart, and pancreas) from mice injected intravenously with AAVhu68.GFP. Samples were prepared and processed as described in Example 4. A positive GFP signal, shown in green, indicates successful transduction of the indicated AAV vectors. Bright field images, shown in black and white, were taken to determine organ morphology, while the corresponding red fluorescent channel was performed as a negative control where applicable.

Fig. 6A shows microscopic images of a representative liver section from mice injected intravenously with AAVhu68.GFP. Positive signal was observed, shown in green.

Fig. 6B shows microscopic images of a representative kidney section from mice injected intravenously with AAVhu68.GFP. Positive signal was observed, shown in green.

Fig. 6C shows microscopic images of a representative section of the heart from mice injected intravenously with AAVhu68.GFP. Positive signal was observed, shown in green.

Fig. 6D shows microscopic images of a representative pancreas section from mice injected intravenously with AAVhu68.GFP. Positive signal was observed, shown in green.

Fig. 7 is an illustration of an intracisternal delivery device including an optional guiding needle for a coaxial route of administration, which includes a 10 cc syringe with a vector, a 10 cc pre-filled flushing syringe, an extension set with a T-shaped connector, a 22G x5 spinal needle, an additional 18G x3.5 guiding needle.

Fig. 8A, B illustrate the production yields of two different AAVhu68 vectors prepared at small scale (Fig. 8A) and very large scale (mega, Fig. 8B) compared to vectors having other capsids. Data for small scale vector preparations were generated using vectors having the AAVhu68, AAV9, AAV8, or AAV8triple capsid and having a vector genome containing the cytomegalovirus promoter (CMV), the firefly luciferase coding sequence, and SV40 poly A (CMV.ffLuciferase.SV40). Mega scale preparations were evaluated using AAVhu68, AAV9, AAV8, or AAV8triple vectors with a vector genome containing the

- 5 048431 CMV motor, intron, immunoadhesin-coding sequence (201Ig IA), and SV40 poly-A.

Fig. 9 shows the purity of production of AAVhu68 vectors produced at mega-scale compared to vectors having other capsids, including AAV8triple, AAV9, and AAV8. Preparations were evaluated using AAVhu68, AAV9, AAV8, or AAV8triple vectors having a vector genome containing the CMV promoter, intron, immunoadhesin-coding sequence (201Ig IA), and SV40 poly A.

Figure 10A, B shows the level of transgene expression of AAVhu68 vectors in male RAG KO mice (n=5/group) injected intramuscularly with either 3/10 ¹¹ GC/mouse (Figure 10A) or 3/10 ¹⁰ GC/mouse (Figure 10B) vector, compared to vectors having other capsids, including AAV8triple, AAV9, and AAV8. The transgene expressed by the rAAV vectors is an immunoadhesin-coding sequence (201Ig IA). The experiment was performed as described in detail in Example 8.

Figure 11A, B shows the level of transgene expression of AAVhu68 vectors in either liver (Figure 11A) or muscle (Figure 11B) of male C57BL/6J mice (n=5/group) injected intramuscularly with 3/10 ¹¹ GC/mouse vector, compared to vectors having other capsids, including AAV8triple, AAV9, and AAV8. The transgene expressed by the rAAV vectors is firefly luciferase. The experiment was performed as described in detail in Example 9.

Figure 12 shows the level of transgene expression of AAVhu68 vectors in male and female cynomolgus monkeys injected intramuscularly with 1/10 ¹³ GC/kg body weight vector, compared to those vectors having other capsids, including AAV8triple, AAV9, and AAV8. The transgene expressed by the rAAV vectors is an immunoadhesin-encoding sequence (201Ig IA). The experiment was performed as described in detail in Example 10.

Detailed description of the invention

Provided herein are nucleic acid and amino acid sequences of a novel isolated adeno-associated virus (AAV), herein referred to as AAVhu68, which is within clade F. AAVhu68 (previously referred to herein as AAV3G2) differs from another clade F virus AAV9 (SEQ ID NO: 5) at two encoded amino acids vp1 at positions 67 and 157, SEQ ID NO: 2. In contrast, another clade F AAV (AAV9, hu31, hu31) has an Ala at position 67 and an Ala at position 157. Provided are novel AAVhu68 capsids and/or engineered AAV capsids having a valine (Val or V) at position 157 based on the numbering of SEQ ID NO: 2, and optionally a glutamic acid (Glu or E) at position 67. In certain embodiments, the protein ratio The ratio of vp3 in the AAVhu68 capsid to the vp1 and vp2 proteins is lower than previously reported for the capsids of AAV9 and other clade F AAVs. In certain embodiments, the AAVhu68 capsid consists of AAVhu68 vpl proteins, AAVhu68 vp2 proteins, and AAVhu68 vp3 proteins in a ratio of about 1 vp1:1 to about 1.5 vp2:3 to about 10 vp3. In certain embodiments of the invention, the rAAVhu68 parent virus or rAAVhu68 population is a composition having an average of about 60 total vp1, vp2, and vp3 proteins in the AAVhu68 capsid, which are present in an average vp1:vp2:vp3 ratio of about 1:about 1:from about 3 to 6. These AAV capsids described herein are useful for generating recombinant AAV vectors (rAAV) that provide good yield and/or packaging efficiency, and for providing rAAV vectors useful for transducing a variety of different cell types and tissues. Such cells and tissue types include, but are not limited to, lung, heart, muscle, liver, pancreas, kidney, brain, hippocampus, motor cortex, cerebellum, nasal epithelial cells, cardiac muscle cells or cardiomyocytes, hepatocytes, lung endothelial cells, myocytes, lung epithelial cells, islet cells, acinar cells, renal cells, and motor neurons.

A recombinant AAV or rAAV is a DNase-resistant viral particle comprising two elements, an AAV capsid and a vector genome comprising at least non-AAV coding sequences packaged within an AAV capsid. Unless otherwise specified, the term may be used interchangeably with the phrase rAAV vector. An rAAV is a replication-defective virus or viral vector because it lacks any functional AAV rep gene or a functional AAV cap gene and is unable to generate progeny. In certain embodiments, the only AAV sequences are AAV inverted terminal repeat (ITR) sequences, typically located at the extreme 5' and 3' ends of the vector genome to allow for gene packaging and regulatory sequences located between the ITRs for packaging within the AAV capsid.

As used herein, the term vector genome refers to a nucleic acid sequence packaged within the rAAV capsid that forms the viral particle. Such nucleic acid sequence comprises AAV inverted terminal repeat (ITR) sequences. In the examples provided herein, the vector genome comprises, at a minimum, in the 5' to 3' direction: an AAV 5' ITR, coding sequence(s), and an AAV 3' ITR. The ITRs may be selected from AAV2, a source of AAV other than the capsid, or other than the full-length

- 6,048,431 meric ITRs. In certain embodiments, the ITRs are derived from the same AAV source as the AAV that provides rep (replication) function during AAV production or trans-complementation. Other ITRs may also be used. Additionally, the vector genome contains regulatory sequences that direct the expression of gene products. Suitable vector genome components are discussed in more detail herein.

rAAVhu68 consists of an AAVhu68 capsid and a vector genome. The AAVhu68 capsid is an aggregate of a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 proteins, and a heterogeneous population of vp3 proteins. As used herein, when used to refer to vp capsid proteins, the term heterogeneous or any grammatical variation thereof refers to a population comprised of elements that are not the same, such as having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO: 2 provides the encoded amino acid sequence of the AAVhu68 vp1 protein.

The AAVhu68 capsid comprises subpopulations of the vp1 proteins, the vp2 proteins, and the vp3 proteins that have modifications from the predicted amino acid residues of SEQ ID NO: 2. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, some subpopulations contain at least one, two, three, or four highly deamidated asparagines (N) positions in the asparagine-glycine pairs of SEQ ID NO: 2 and optionally further contain other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications. SEQ ID NO: 14 provides the amino acid sequence of a modified AAVhu68 capsid illustrating positions that may have some percentage of deamidated or otherwise modified amino acids. Various combinations of these and other modifications are described herein.

As used herein, the term "vp protein subpopulation" refers to a group of vp proteins that share at least one defined characteristic in common and that consists of at least one member of the group to less than all members of a reference group, unless otherwise specified. For example, a vp1 protein subpopulation is at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid, unless otherwise specified. A vp3 protein subpopulation may be at least one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, vp1 proteins may be a subpopulation of vp proteins; vp2 proteins may be a distinct subpopulation of vp proteins, and vp3 proteins may be yet another additional subpopulation of vp proteins in an assembled AAV capsid. In another example, the vp1, vp2, and vp3 proteins may comprise subpopulations having different modifications, such as at least one, two, three, or four highly deamidated asparagines, such as in asparagine-glycine pairs.

Unless otherwise specified, the term "highly deamidated" refers to at least 45% deamidation, at least 50% deamidation, at least 60% deamidation, at least 65% deamidation, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidation at a reference amino acid position compared to a predicted amino acid sequence at the reference amino acid position (e.g., at least 80% of the asparagines at amino acid 57 of SEQ ID NO: 2 may be deamidated based on the total number of vp1 proteins, or 20% of the asparagines at amino acid 409 of SEQ ID NO: 2 may be deamidated based on the total number of proteins vp1, vp2 and vp3). Such percentages may be determined using 2D gel electrophoresis analysis, mass spectrometry methods or other suitable methods.

Without wishing to be bound by theory, it is believed that deamidation of at least the highly deamidated residues in the vp proteins in the AAVhu68 capsid is largely non-enzymatic in nature, caused by functional groups in the capsid protein that deamidate select asparagines and, to a lesser extent, glutamine residues. Efficient capsid assembly from the majority of deamidated vp1 proteins suggests that either these events occur after capsid assembly or that deamidation in individual monomers (vp1, vp2, or vp3) is structurally well tolerated and does not significantly affect assembly dynamics. Extensive deamidation in the VP1 unique region (VP1-u) (~aa 1-137), which is generally thought to be located internally before cell entry, suggests that VP deamidation may occur prior to capsid assembly.

Without wishing to be bound by any theory, deamidation of N may occur via the nitrogen atom of the C-terminal residue of its backbone, resulting in a nucleophilic attack on the carbon atom of the amide group of the side chain of Asn. It is believed that an intermediate succinimide residue with a closed ring is formed. The succinimide residue then undergoes rapid hydrolysis, resulting in the final product aspartic acid (Asp) or isoaspartic acid (IsoAsp). Therefore, in certain embodiments, deamidation of asparagine (N or Asn) results in Asp or IsoAsp, which can be interconverted via a succinimide intermediate, for example as shown below.

- 7 048431 o

O

Isoaspartic acid

As provided herein, each deamidated N of SEQ ID NO: 2 can independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartate, and/or an interconvertible mixture of Asp and isoAsp, or combinations thereof. Any suitable ratio of α- to isoaspartic acid can be present. For example, in certain embodiments, the ratio can be from 10:1 to 1:10 aspartic to isoaspartic, about 50:50 aspartic:isoaspartic, or about 1:3 aspartic:isoaspartic, or another selected ratio.

In certain embodiments, one or more glutamines (Q) in SEQ ID NO: 2 are deamidated to glutamic acid (Glu), i.e., α-glutamic acid, γ-glutamic acid (Glu), or a mixture of α- and γ-glutamic acids that can be interconverted via a common glutarinimide intermediate. Any suitable ratio of α- and γ-glutamic acid can be present. For example, in certain embodiments, the ratio can be from 10:1 to 1:10 α to γ, about 50:50 α:γ, or about 1:3 α:γ, or another selected ratio.

glutamic acid (a-Glu)

isoglutamic acid (γ-Glu)

Thus, rAAVhu68 includes subpopulations in the rAAVhu68 capsid of the vp1, vp2 and/or vp3 proteins with deamidated amino acids, including at least at least one subpopulation containing at least one highly deamidated asparagine. In addition, other modifications may include isomerization, especially at selected positions of aspartic acid residues (D or Asp). In still other embodiments, modifications may include amidation at the Asp position.

In certain embodiments, the AAVhu68 capsid comprises vp1, vp2, and vp3 subpopulations having at least 4 to at least about 25 deamidated amino acid residue positions, of which at least 1 to 10% are deamidated compared to the encoded amino acid sequence of SEQ ID NO: 2. The majority of these may be N residues. However, Q residues may also be deamidated.

In certain embodiments, the AAV68 capsid is further characterized by one or more of the following. The AAV hu68 capsid proteins comprise: AAVhu68 vp1 proteins produced by expressing a nucleic acid sequence that encodes the predicted amino acid sequence from 1 to 736 of SEQ ID NO: 2, vp1 proteins produced from SEQ ID NO: 1, or vp1 proteins produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 1 that encodes the predicted amino acid sequence from 1 to 736

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SEQ ID NO:2; AAVhu68 vp2 proteins produced by expressing a nucleic acid sequence that encodes a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 2, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO: 1, or vp2 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO: 1 that encodes a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 2, and/or AAVhu68 vp3 proteins produced by expressing a nucleic acid sequence that encodes a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO: 2, vp3 proteins produced from a sequence comprising at least nucleotides from 607 to 2211 of SEQ ID NO: 1, or vp3 proteins obtained from a nucleic acid sequence at least 70% identical to at least nucleotides from 607 to 2211 of SEQ ID NO: 1, which encodes a predicted amino acid sequence of at least about amino acids from 203 to 736 of SEQ ID NO: 2.

Additionally or alternatively, there is provided an AAV capsid that comprises a heterogeneous population of vp1 proteins optionally comprising a valine at position 157, a heterogeneous population of vp2 proteins optionally comprising a valine at position 157, and a heterogeneous population of vp3 proteins, wherein at least subpopulations of the vp1 and vp2 proteins comprise a valine at position 157 and optionally further comprise a glutamic acid at position 67 based on the capsid vp1 numbering of SEQ ID NO: 2. Additionally or alternatively, there is provided an AAVhu68 capsid that comprises a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 2, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO: 2, wherein the vp1, vp2 and vp3 proteins comprise subpopulations with amino acid modifications.

The vp1, vp2, and vp3 proteins of AAVhu68 are typically expressed as alternative splice variants encoded by the same nucleic acid sequence that encodes the full-length vp1 amino acid sequence SEQ ID NO: 2 (amino acids 1 to 736). Optionally, the vp1 coding sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, the sequence may be co-expressed with one or more nucleic acid sequences that encode the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 2 (about aa 203 to 736) without the xp1 unique region (about aa 1 to about aa 137) and/or without the xp2 unique regions (about aa 1 to about aa 202), or a strand complementary thereto corresponding to the mRNA or tRNA (from about nucleotide 607 to about nucleotide 2211 of SEQ ID NO: 1), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1, which encodes aa 203 to 736 of SEQ ID NO: 2. Additionally or alternatively, the xp1 and/or vp2 coding sequence can be co-expressed with a nucleic acid sequence that encodes the vp2 amino acid sequence of AAVhu68 of SEQ ID NO: 2 (about aa 138 to 736) without the vp1 unique region (about aa 1 to about 137), or the complementary strand thereof corresponding to the mRNA or tRNA (nucleotide 412 to 22121 of SEQ ID NO: 1), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1, which encodes about aa from 138 to 736 SEQ ID NO: 2.

As described herein, rAAVhu68 has a rAAVhu68 capsid produced in a production system that expresses capsids from AAVhu68 nucleic acid that encodes the vp1 amino acid sequence of SEQ ID NO: 2, and optionally additional nucleic acid sequences, such as encoding a vp3 protein free of regions unique to vp1 and/or vp2.

rAAVhu68, produced using a single vp1 nucleic acid sequence, produces heterogeneous populations of vp1 proteins, vp2 proteins, and vp3 proteins. Specifically, the AAVhu68 capsid contains subpopulations of vp1 proteins, vp2 proteins, and vp3 proteins that have modifications from the predicted amino acid residues in SEQ ID NO: 2. These subpopulations include, at a minimum, deamidated asparagine residues (N or Asn). For example, asparagines in asparagine-glycine pairs are highly deamidated.

In one embodiment of the invention, the vp1 nucleic acid sequence of AAVhu68 comprises the sequence of SEQ ID NO: 1, or a strand complementary thereto, such as the corresponding mRNA or tRNA. In some embodiments of the invention, the vp2 and/or vp3 proteins may be additionally or alternatively expressed from nucleic acid sequences,

- 9 048 431 different from vpl sequences, for example to change the ratio of vp proteins in a selected expression system. In some embodiments, the invention also provides a nucleic acid sequence that encodes the amino acid sequence of vp3 AAVhu68 SEQ ID NO: 2 (about aa 203 to 736) without the \p1 unique region (from about aa 1 to about aa 137) and/or without the \p2 unique regions (from about aa 1 to about aa 202), or a complementary strand corresponding to mRNA or tRNA (from about nucleotide 607 to about nucleotide 2211 of SEQ ID NO: 1). In some embodiments, the invention also provides a nucleic acid sequence that encodes the amino acid sequence of vp2 AAVhu68 SEQ ID NO: 2 (about aa 138 to 736) without the ur1-unique region (from about aa 1 to about 137), or the complementary strand thereof, corresponding to the mRNA or tRNA (nucleotide 412 to 2211 of SEQ ID NO: 1).

However, other nucleic acid sequences that encode the amino acid sequence of SEQ ID NO: 2 may be selected for use in producing rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 1, or a sequence that is at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1, which encodes SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 1, or a sequence that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical from about nucleotide 412 to about nucleotide 2211 SEQ ID NO: 1, which encodes the vp2 capsid protein (about aa from 138 to 736) of SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence comprises a nucleic acid sequence from about nucleotide 607 to about nucleotide 2211 of SEQ ID NO: 1, or a sequence that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleotides of SEQ ID NO: 1, which encodes the vp3 capsid protein (about aa from 203 to 736) of SEQ ID NO: 2.

One skilled in the art can design nucleic acid sequences encoding the AAVhu68 capsid, including DNA (genomic or cDNA) or RNA (e.g., mRNA). In certain embodiments, the nucleic acid sequence encoding the AAVhu68 capsid protein vp1 is provided in SEQ ID NO: 1. See also Figs. 1B-1D. In other embodiments, a nucleic acid sequence that is 70% to 99.9% identical to SEQ ID NO: 1 can be selected to express the AAVhu68 capsid proteins. In certain other embodiments, the nucleic acid sequence is at least about 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% to 99.9% identical to SEQ ID NO: 1. Such nucleic acid sequences can be codon optimized for expression in a selected system (i.e., cell type), which can be constructed in a variety of ways. This optimization can be performed using methods that are available online (e.g., GeneArt), published methods, or from a company that provides codon optimization services, such as DNA2.0 (Menlo Park, Calif., USA). One method for codon optimization is described, for example, in U.S. International Patent Publication No. WO 2015/012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. Patent Publication No. 2014/0032186 and U.S. Patent Publication No. 2006/0136184. Accordingly, the entire length of the open reading frame (ORF) is modified for the product. However, in some embodiments, only a portion of the ORF may be modified. By applying one of these methods to any given polypeptide sequence, frequencies can be used to obtain a nucleotide fragment of a codon-optimized coding region that encodes the polypeptide. A number of options are available for making the actual codon changes or for synthesizing codon-optimized coding regions designed in accordance with the method described herein. Such modifications or synthesis may be accomplished by standard and routine molecular biology manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs, each 80-90 nucleotides in length and spanning the length of the desired sequence, are synthesized using standard techniques. These oligonucleotide pairs are synthesized such that when annealed, they form double-stranded fragments of 80-90 base pairs containing sticky ends, e.g., each oligonucleotide in a pair is synthesized with an extension of 3, 4, 5, 6, 7, 8, 9, 10 or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each oligonucleotide pair are designed to anneal with a single

- 10 048431 with the bud end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal and then about five or six of these double-stranded fragments are allowed to anneal together via the sticky single-stranded ends, then they are co-ligated and cloned into a standard bacterial cloning vector, for example the TOPO® vector available from Invitrogen Corporation, Carlsbad, CA, USA. The construct is then sequenced by standard techniques. Several of these constructs, containing 5-6 fragments of co-ligated 80-90 bp fragments, i.e. fragments of about 500 bp, are prepared so that the entire desired sequence is represented by a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and co-ligated to form the final construct. The completed construct is then cloned into a standard bacterial cloning vector and sequenced. Additional techniques will be apparent to those skilled in the art. Gene synthesis is also commercially available.

In some embodiments, the asparagine (N) in the N-G pairs of the AAVhu68 vp1, vp2, and vp3 proteins is extensively deamidated. In certain embodiments, the AAVhu68 capsid comprises subpopulations of the AAV vp1, vp2, and/or vp3 capsid proteins having at least four asparagine (N) positions in the AAVhu68 capsid proteins that are extensively deamidated. In some embodiments, about 20% to 50% of the N-N pairs (excluding N-N-N triplets) exhibit deamidation. In certain embodiments, the first N is deamidated. In certain embodiments, the second N is deamidated. In certain embodiments, the deamidation is between about 15% to about 25% deamidation. Deamidation at Q at position 259 of SEQ ID NO: 2 accounts for about 8% to about 42% of the AAVhu68 vp1, vp2, and vp3 capsid proteins of the AAVhu68 protein.

In certain embodiments, the rAAVhu68 capsid is further characterized by amidation at D297 of the vp1, vp2, and vp3 proteins. In some embodiments, about 70% to about 75% of the D at position 297 of the vp1, vp2, and/or vp3 proteins in the AAVhu68 capsid are amidated, based on the numbering of SEQ ID NO: 2.

In certain embodiments, at least one Asp in the vp1, vp2 and/or vp3 capsid isomerizes to D-Asp. Such isomers are typically present in amounts of less than about 1% Asp at one or more positions of residues 97, 107, 384, based on the numbering of SEQ ID NO: 2.

In some embodiments, rAAVhu68 has an AAVhu68 capsid comprising vp1, vp2, and vp3 proteins with subpopulations comprising combinations of two, three, four, or more deamidated residues at the positions indicated in the table below. Deamidation in rAAV can be determined using 2D gel electrophoresis and/or mass spectrometry and/or protein modeling methods. On-line chromatography can be performed with an Acclaim PepMap column and a Thermo UltiMate 3000 RSLC system (Thermo Fisher Scientific) coupled to a Q Exactive HF with a NanoFlex source (Thermo Fisher Scientific). MS data are obtained using a top 20 ion data dependent method for Q Exactive HF, dynamically selecting the most abundant, as yet unsequenced precursor ions from scans (200-2000 m/z). Sequencing, performed by high-energy impact dissociation fragmentation with a target of 1e5 ions determined by predictive automatic gain control and precursor extraction, was performed with a window of 4 m/z. Scan views were acquired at a resolution of 120,000 at m/z 200. The resolution for the HCD spectra could be set to 30,000 at m/z 200 with a maximum ion injection time of 50 ms and a normalized collision energy of 30. The RF S-objective level could be set to 50 to ensure optimal transmission of the m/z region occupied by protease-derived peptides. Precursor ions with single, unassigned, or six or higher charge states could be excluded from fragmentation selection. BioPharma Finder 1.0 software (Thermo Fischer Scientific) could be used to analyze the acquired data. For peptide mapping, the search is performed using a single-entry FASTA protein database with carbamidomethylation set as a fixed modification; and oxidation, deamidation, and phosphorylation set as variable modifications. A mass accuracy of 10 ppm, high protease specificity, and a confidence level of 0.8 are set for the MS/MS spectra. Examples of suitable proteases may include, for example, trypsin or chymotrypsin. Mass spectrometric identification of deamidated peptides is relatively straightforward since deamidation increases the mass of the intact molecule by +0.984 Da (the mass difference between the -OH and -NH2 groups). The percentage of deamidation of a particular peptide is determined by the mass area of the deamidated peptide divided by the sum of the areas of the deamidated and native peptides. Given the number of possible deamidation sites, isobaric species that are deamidated at different locations may co-migrate in the same peak. Therefore, fragment ions derived from peptides with multiple potential deamidation sites can be used to

- 11 048431 determining the location or differentiation of multiple deamidation sites. In these cases, the relative intensities within the observed isotopic patterns can be used to specifically determine the relative abundance of the various deamidated peptide isomers. This method assumes that the fragmentation efficiency is the same for all isomeric types and is independent of the deamidation site. One skilled in the art will appreciate that a number of variations of these illustrative methods can be used. For example, suitable mass spectrometers can include, for example, a quadrupole time-of-flight (QTOF) mass spectrometer such as a Waters Xevo or an Agilent 6530, or an orbitrap instrument such as an Orbitrap Fusion or Orbitrap Velos (Thermo Fisher). Suitable liquid chromatography systems include, for example, an Acquity UPLC system from Waters or Agilent systems (1100 or 1200 series). Suitable data analysis software may include, for example, MassLynx (Waters), Pinpoint and Pepfinder (Thermo Fischer Scientific), Mascot (Matrix Science), Peaks DB (Bioinformatics Solutions). Other methods may be described, for example, in X. Jin et al., Hu Gene Therapy Methods, Vol. 28, No. 5, pp. 255267, published online June 16, 2017.

Deamidation Based on the predicted AAVHu68 [SEQ ID NO: 2] Average % of VP1/VP2/VP3 proteins in AAVhu68 capsid Deamidated residue+1 (adjacent AA) Wide range of percentages (%) Narrow ranges (%) N57 (N-G) from 78 to 100% from 80 to 100, from 85 to 97 N66 (N-E) from 0 to 5 0, from 1 to 5 N94 (N-H) from 0 to 15, 0, 1 to 15, 5 to 12, 8 N113 (N-L) from 0 to 2 0, from 1 to 2 -N253 (N-N) from 10 to 25 from 15 to 22 Q259 (Q-D from 8 to 42 from 10 to 40, from 20 to 35 -N270 from 12 to 30 from 15 to 28

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(N-D) -N304 (N-N) (position 303 is also N) from 0 to 5 from 1 to 4 N319 (N-I) from 0 to 5 0, 1 to 5, 1 to 3 N329 * (N-G)*(position 328 is also N) from 65 to 100 from 70 to 95, from 85 to 95, from 80 to 100, from 85 to 100, N336 (N-N) from 0 to 100 0, 1 to 10, 25 to 100, 30 to 100, 30 to 95 -N409 (N-N) from 15 to 30 from 20 to 25 N452 (N-G) from 75 to 100 from 80 to 100, from 90 to 100, from 95 to 100, N477 (N-Y) from 0 to 8 0, from 1 to 5 N512 (N-G) from 65 to 100 from 70 to 95, from 85 to 95, from 80 to 100, from 85 to 100, -N515 (N-S) from 0 to 25 0, from 1 to 10, from 5 to 25, from 15 to 25 -Q599 (Asn-Q-Gly) from 1 to 20 from 2 to 20, from 5 to 15 N628 (N-F) from 0 to 10 0, 1 to 10, 2 to 8 N651 (N-T) from 0 to 3 0, from 1 to 3 N663 (N-K) from 0 to 5 0, 1 to 5, 2 to 4 N709 (N-N) from 0 to 25 0, 1 to 22, 15 to 25 N735 from 0 to 40 0. from 1 to 35, from 5 to 50, from 20 to 35

In certain embodiments, the AAVhu68 capsid is characterized by having capsid proteins in which at least 45% of the N-residues are deamidated at at least one of positions N57, N329, N452, and/or N512, based on the amino acid sequence numbering of SEQ ID NO: 2. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N-residues at one or more of these N-G positions (i.e., N57, N329, N452, and/or N512, based on the amino acid sequence numbering of SEQ ID NO: 2) are deamidated. In these and other embodiments, the AAVhu68 capsid is further characterized by having a population of proteins in which about 1% to about 20% of the N-residues have deamidation at one or more of positions N94, N253, N270, N304, N409, N477, and/or Q599, based on the amino acid sequence numbering of SEQ ID NO: 2. In some embodiments, AAVhu68 comprises at least a subpopulation of the vp1, vp2, and/or vp3 proteins that are deamidated at one or more of positions N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, based on the numbering of the amino acid sequence of SEQ ID NO: 2, or a combination thereof. In some embodiments, the capsid proteins may have one or more amidated amino acids.

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Other modifications are also observed, most of which do not result in the conversion of one amino acid residue to another amino acid residue. Optionally, at least one Lys in the vp1, vp2 and vp3 capsid is acetylated. Optionally, at least one Asp in the vp1, vp2 and/or vp3 capsid isomerizes to D-Asp. Optionally, at least one S (Ser, Ser) in the vp1, vp2 and/or vp3 capsid is phosphorylated. Optionally, at least one T (Thr, threonine) in the vp1, vp2 and/or vp3 capsid is phosphorylated. Optionally, at least one W (Trp, tryptophan) in the vp1, vp2 and/or vp3 capsid is oxidized. Optionally, at least one M (Met, methionine) in the vp1, vp2 and/or vp3 capsid is oxidized. In certain embodiments, the capsid proteins have one or more phosphorylations. For example, some vp1 capsid proteins may be phosphorylated at position 149.

In certain embodiments, the AAVhu68 capsid comprises a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, wherein the vp1 proteins comprise a glutamic acid (Glu) at position 67 and/or a valine (Val) at position 157; a heterogeneous population of vp2 proteins, optionally comprising a valine (Val) at position 157; and a heterogeneous population of vp3 proteins. The AAVhu68 capsid comprises at least one subpopulation in which at least 65% of the asparagines (N) in the asparagine-glycine pairs located at position 57 of the vp1 proteins and at least 70% of the asparagines (N) in the asparagine-glycine pairs at positions 329, 452, and/or 512 of the vp1, v2, and vp3 proteins are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO: 2, wherein the deamidation results in a change in amino acid. As discussed in more detail herein, the deamidated asparagines can be deamidated to aspartic acid, isoaspartic acid, an aspartic acid/isoaspartic acid interconvertible pair, or combinations thereof. In certain embodiments, rAAVhu68 is further characterized by one or more of the following: (a) each of the vp2 proteins is independently the product of a nucleic acid sequence encoding at least a vp2 protein of SEQ ID NO: 2; (b) each of the vp3 proteins is independently the product of a nucleic acid sequence encoding at least a vp3 protein of SEQ ID NO: 2; (c) the nucleic acid sequence encoding the vp1 proteins is SEQ ID NO: 1, or a sequence that is at least 70% to at least 99% (such as at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2. Optionally, the sequence is used separately to express the vp1, vp2, and vp3 proteins. Alternatively, the sequence may be co-expressed with one or more nucleic acid sequences that encode the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 2 (about aa 203 to 736) without the vp1 unique region (about aa 1 to about aa 137) and/or without the xp2 unique regions (about aa 1 to about aa 202), or a strand complementary thereto corresponding to an mRNA or tRNA (from about nucleotide 607 to about nucleotide 2211 of SEQ ID NO: 1), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1, which encodes aa 203 to 736 of SEQ ID NO: 2. Additionally or alternatively, the vp1 and/or vp2 coding sequence can be co-expressed with a nucleic acid sequence that encodes the vp2 amino acid sequence of AAVhu68 of SEQ ID NO: 2 (about aa 138 to 736) without the xp1 unique region (about aa 1 to about 137), or the complementary strand thereof corresponding to the mRNA or tRNA (nucleotide 412 to 2211 of SEQ ID NO: 1), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1, which encodes about aa 138 to 736 SEQ ID NO: 2.

Additionally or alternatively, the rAAVhu68 capsid comprises at least a subset of the vp1, vp2 and/or vp3 proteins that are deamidated at one or more positions N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N512, N515, N598, Q599, N628, N651, N663, N709, based on the numbering of SEQ ID NO: 2, or combinations thereof; (e) the rAAVhu68 capsid comprises a subpopulation of vp1, vp2 and/or vp3 proteins that contain from 1% to 20% deamidation at one or more of positions N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N336, N409, N410, N477, N515, N598, Q599, N628, N651, N663, N709, based on the numbering of SEQ ID NO: 2, or combinations thereof; (f) the rAAVhu68 capsid comprises a vp1 subpopulation in which from 65% to 100% of the N at position 57 of the vp1 proteins, based on the numbering of SEQ ID NO: 2, is deamidated; (g) the rAAVhu68 capsid comprises a subpopulation of vp1 proteins in which between 75% and 100% of the N at position 57 of the vp1 proteins is deamidated; (h) the rAAVhu68 capsid comprises a subpopulation of vp1 proteins, vp2 proteins and/or vp3 proteins in which between 80% and 100% of the N at position 329, based on the numbering of SEQ ID NO: 2, is deamidated; (i) the rAAVhu68 capsid comprises a subpopulation of vp1 proteins, vp2 proteins and/or vp3 proteins in which between 80% and 100% of the N at position 452, based on the numbering of SEQ ID NO:

- 14 048431

2, deamidated; (j) the rAAVhu68 capsid comprises a subpopulation of vpl proteins, vp2 proteins, and/or vp3 proteins in which 80% to 100% of the N at position 512, based on the numbering of SEQ ID NO: 2, is deamidated; (k) the rAAV comprises a total of about 60 capsid proteins in a ratio of about 1 vp1 to about 1 to 1.5 vp2 to 3 to 10 vp3 proteins; (l) the rAAV comprises a total of about 60 capsid proteins in a ratio of about 1 vp1 to about 1 vp2 to 3 to 9 vp3 proteins.

In certain embodiments, AAVhu68 is modified to alter the glycine in the asparagine-glycine pair to reduce deamidation. In other embodiments, asparagine is replaced with another amino acid, such as glutamine, which is deamidated at a lower rate; or an amino acid that lacks amide groups (e.g., glutamine and asparagine contain amide groups); and/or an amino acid that lacks amino groups (e.g., lysine, arginine, and histidine contain amide groups). As used herein, amino acids that lack amide or amine side groups include, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cystine, phenylalanine, tyrosine, or tryptophan and/or proline. Modifications such as those described may be in one, two, or three asparagine-glycine pairs found in the encoded amino acid sequence of AAVhu68. In some embodiments, such modifications are not made in all four asparagine-glycine pairs. Thus, a method is provided for reducing deamidation of AAVhu68 and/or engineered variants of AAVhu68 having lower rates of deamidation. Additionally or alternatively, one or more other amide amino acids may be replaced with a non-amide amino acid to reduce deamidation of AAVhu68.

These amino acid modifications can be made by conventional genetic engineering techniques. For example, a nucleic acid sequence comprising modified vp codons of AAVhu68 can be created in which one to three codons encoding glycine at positions 58, 330, 453 and/or 513 in SEQ ID NO: 2 (arginine-glycine pair) are modified to encode amino acids other than glycine. In certain embodiments, a nucleic acid sequence comprising modified arginine codons can be engineered with one to three arginine-glycine pairs located at positions 57, 329, 452 and/or 512 in SEQ ID NO: 2 such that the modified codon encodes an amino acid other than arginine. Each modified codon can encode different amino acids. Alternatively, one or more altered codons can encode the same amino acid. In some embodiments, these modified AAVhu68 nucleic acid sequences can be used to produce a mutant rAAVhu68 having a capsid with lower deamination than the native hu68 capsid. Such a mutant rAAVhu68 may have reduced immunogenicity and/or increased storage stability, particularly when stored in suspension form. As used herein, the term codon refers to three nucleotides in a sequence that encodes an amino acid.

As used herein, the term encoded amino acid sequence refers to an amino acid that is predicted based on the translation of a known DNA codon of a reference nucleic acid sequence that is translated into an amino acid. The following table illustrates DNA codons and twenty common amino acids, showing both the single-letter code (SLC) and the three-letter code (3LC).

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Amino acid SLC 3 LC DNA codons Isoleucine I Not ATT, ATC, ATΑ Leucine L Leu CTT, CTC, STA, CTG, TTA, TTG Valin V Vai GTT, GTC, GTA, GTG Phenylalanine F Phe TTT, TTS Methionine M Met ATG Cysteine WITH Cys TGT, TGC Alanine A Ala GCT, GCC, GCA, GCG Glycine G Gly GGT, GGC, GGA, GGG Proline R Pro CCT, CCC, CCA, CCG Threonine T Thr ACT, ACC, ACA, ACG Serine S Ser TCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y Tyr TAT, TAS Tryptophan W Trp TGG Glutamine Q Gin SAA, CAG

Asparagine N Asn AAT, AAC G istidine N His CAT, CAC Glutamic acid E Glu GAA, GAG Aspartic acid D Asp GAT, GAC Lysine TO Lys AAA, AAG Arginine R Arg CGT, CGC, CGA, CGG, AGA, AGG Stop codons Stop TAA, TAG, TGA

In some embodiments, AAVhu68 capsids may be used. For example, such capsids may be used to generate monoclonal antibodies and/or to generate reagents useful in assays for monitoring AAVhu68 concentration levels in patients receiving gene therapy. Methods for producing useful antibodies against AAVhu68, labeling such antibodies or empty capsids, and suitable assay formats are known to those skilled in the art.

In certain embodiments, provided herein is a nucleic acid sequence of SEQ ID NO: 1 or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% of a sequence that encodes the vp1 amino acid sequence of SEQ ID NO: 2 with a modification (e.g., a deamidated amino acid) as described herein. In certain embodiments, the vp1 amino acid sequence is reproduced in SEQ ID NO: 14.

As used herein, the term clade, as it pertains to AAV groups, refers to groups of AAV that are phylogenetically related to each other, as determined using the Neighbor-Joining algorithm with a bootstrap value of at least 75% (from at least 1000 replicates) and a Poisson correction distance of no more than 0.05, based on an alignment of the AAV vp1 amino acid sequence. The Neighbor-Joining algorithm has been described in the literature. See, for example, M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this

- 16 048431 algorithm. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, as well as the sequence of the AAV capsid protein vp1, one skilled in the art can easily determine whether a selected AAV is contained in one of the clades identified herein, in another clade, or outside these clades. See, for example, G Gao, et al, J Virol, 2004 Jun; 78(10: 6381-6388, which identifies clades A, B, C, D, E, and F and provides nucleic acid sequences of a novel AAV, GenBank accession numbers AY530553 to AY530629. See also WO 2005/033321.

In one embodiment, the invention provides an engineered molecule comprising a spacer sequence between the coding sequence of vp1 of AAVhu68 and the coding sequences of the AAVhu68 repeat. This coding sequence is: atgacttaaaccaggt, SEQ ID NO: 9. The coding sequence for rep52 of AAVhu68 is reproduced in SEQ ID NO: 3. The protein sequence of rep52 is reproduced in SEQ ID NO: 4.

In one embodiment, a method is provided for increasing the yield of rAAV and thereby increasing the amount of rAAV that is present in the supernatant fluid prior to or without the need for cell lysis. This method involves engineering the AAV UL capsid gene to express a capsid protein having a Glu at position 67 rather than a Val at position 157, based on alignment with the amino acid numbering of the vp1 capsid protein of AAVhu68. In other embodiments, the method involves engineering the VP1 capsid gene of AAVhu68 to express a capsid protein having a Val at position 157 rather than a Glu at position 67. Such other AAV may conveniently be selected from another AAV clade F, or an AAV in clade A, B, C, D, or E. In certain embodiments, the AAV is selected from clade C, D, E, or F. In other embodiments, the AAV is selected from clade C, D, or E.

In other embodiments, the method comprises increasing the yield of rAAV and thereby increasing the amount of rAAV that is present in the supernatant fluid prior to or without the need for cell lysis. The method comprises engineering the AAV UL capsid gene to express a capsid protein having a Glu at position 67, a Val at position 157, or both based on the alignment with the amino acid numbering of the AAVhu68 vp1 capsid protein. In other embodiments, the method comprises engineering the VP2 capsid gene to express a capsid protein having a Val at position 157. In other embodiments, the rAAV has a modified capsid comprising both the Glu at position 67 and the Val at position 157 variants of the vp1 and vp2 capsid proteins.

In other embodiments, AAVhu68 may be engineered to have Ser, Gly, Ser, or Thr at position 67, according to vp1 numbering [SEQ ID NO: 2], while maintaining Val at position 157. In yet further embodiments, AAVhu68 may be engineered to have He or Leu at position 157, according to vp1 numbering [SEQ ID NO: 2]. In yet another embodiment, AAVhu68 may be engineered to have Ser, Gly, Ser, or Thr at position 67 and He or Leu at position 157, according to vp1 numbering [SEQ ID NO: 2].

In another embodiment, the invention provides a method of packaging a transgene into a clade F AAV that provides at least a 15% increase in the yield of the packaged vector compared to AAV9, said method comprising: culturing a host cell culture according to suitable conditions. In some embodiments, the increase is an increase in yield by at least 90%. In other embodiments, the increase is an increase in yield by at least 200%.

In a comparison between AAVhu68 and AAVrh10, AAVhu68 was found to provide better transduction efficiency than AAVrh10 at a low dose (e.g., about 1x 109 GC) following intracerebroventricular administration. In another comparison between AAVhu68 and AAV9, AAVhu68 was found to provide better transduction efficiency than AAV9 in the cerebellum, motor cortex, and hippocampus of the brain (e.g., at about 1x1011 GC) following intracerebroventricular administration.

In some embodiments, the present invention provides an AAVhu68 vector comprising a vector genome that expresses an antibody directed against the HER2 receptor. Such a vector is useful for treating and/or preventing cancer.

As used herein, the term AAV9 capsid is a self-assembling AAV capsid consisting of a plurality of AAV9 vp proteins. The AAV9 vp proteins are typically expressed as alternative splice variants encoded by the nucleic acid sequence of SEQ ID NO: 5 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% similar thereto, which encodes the vp1 amino acid sequence of SEQ ID NO: 6 (GenBank Accession: AAS99264). These splice variants result in proteins of varying lengths of SEQ ID NO: 6. In some embodiments, the AAV9 capsid comprises an AAV having an amino acid sequence that is 99% identical to AAS99264 or

- 17 048431

99% identical to SEQ ID NO: 6. See also US7906111 WO 2005/033321. AAV9 variants used herein include those described in, for example, WO2016/049230, US8927514, US2015/0344911, and US8734809.

Methods for producing the capsid, its coding sequences, and methods for producing rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081–6086 (2003) and US 2013/0045186 A1.

The term substantial homology or significant similarity, when referring to a nucleic acid or a fragment thereof, indicates that, when optimally aligned with corresponding nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity between at least about 95% to 99% of the aligned sequences. Preferably, the homology is in the full-length sequence, or an open reading frame thereof, or another suitable fragment that is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

The term sequence identity, percent sequence identity or percent identity in the context of nucleic acid sequences refers to residues in two sequences that are similar when aligned for maximum match. The comparison of sequence identities can be performed over the entire length of the genome, the entire length of the gene encoding sequence or a fragment of at least about 500 to 5000 nucleotides is desirable. However, identity between smaller fragments may also be necessary, for example, at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. Likewise, percent sequence identity can be readily determined for amino acid sequences, over the entire length of a protein or fragment thereof. Accordingly, a fragment is at least 8 amino acids in length and can be up to about 700 amino acids. Examples of suitable fragments are described herein.

The term substantial homology or significant similarity when referring to amino acids or fragments thereof indicates that, when optimally aligned with appropriate insertions or deletions of amino acids with another amino acid (or its complementary chain), there is amino acid sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homology is distributed over the entire sequence or a protein thereof, such as a cap protein, a rep protein, or a fragment thereof, which is at least 8 amino acids long, or more preferably at least 15 amino acids long. Examples of suitable fragments are described herein.

By the term highly conservative is meant at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. The identity is easily determined by a person skilled in the art, by referring to algorithms and computer programs known to those skilled in the art.

Typically, when identity, homology, or similarity between two different adeno-associated viruses is discussed, the identity, homology, or similarity is determined with respect to aligned sequences. Aligned sequences or alignments refer to a plurality of nucleotide sequences or protein (amino acid) sequences, often containing corrections for missing or additional bases or amino acids compared to a reference sequence. In the examples, AAV alignments are performed using the published AAV9 sequences as a reference. Alignments are performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Examples of such programs include Clustal Omega, Clustal W, CAP Sequence Assembly, MAP, and MEME, which can be accessed via web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, Vector NTI utilities are also used. A number of algorithms are also known in the art that can be used to measure the identity of nucleotide sequences, including those included in the programs described above. As another example, polynucleotide sequences can be compared using the Fasta™ program in GCG version 6.1. Fasta™ provides alignments and percent sequence identity of the regions of best overlap between a query and target sequence. For example, the percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (word size of 6 and NOPAM factor for the scoring matrix), as provided in GCG version 6.1, incorporated herein by reference. Multiple sequence alignment programs are also available for amino acid sequences, such as the Clustal Omega, Clustal X, MAP, PIMA, MSA, BLOCKMAKER, MEME, and Match-Box programs. In general, any

- 18 048431 of these programs are used with default settings, although one skilled in the art can change these settings if necessary. Alternatively, one skilled in the art can use another algorithm or computer program that provides at least the same level of identity or alignment as provided by these algorithms and programs. See, for example, J. D. Thomson et al, Nucl. Acids. Res., A comprehensive comparison of multiple sequence alignments, 27(13):2682-2690 (1999).

I. rAAV vectors.

As noted above, the novel AAVhu68 sequences and proteins are useful in the production of rAAV, and are also useful in the production of recombinant AAV vectors, which can be antisense delivery vectors, gene therapy vectors, or vaccine vectors. In addition, the engineered AAV capsids described herein, such as those having mutant amino acids at position 67, 157, or both positions relative to the numbering of the vp1 capsid protein in SEQ ID NO: 2, can be used to engineer rAAV vectors for the delivery of a variety of suitable nucleic acid molecules to target cells and tissues.

The genomic sequences that are packaged into the AAV capsid and delivered into the host cell typically consist of, at a minimum, a transgene and its regulatory sequences and AAV inverted terminal repeats (ITRs). Both single-stranded AAV and self-complementary (sc) AAV are encompassed by rAAV. The transgene is a coding nucleic acid sequence, heterologous to the vector sequence, that encodes a polypeptide, protein, functional RNA molecule (e.g., microRNA, microRNA inhibitor), or other gene product of interest. The coding nucleic acid sequence is operably linked to regulatory components in a manner that allows for transcription, translation, and/or expression of the transgene in a cell of the target tissue.

AAV vector sequences typically contain cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., B. J. Carter, in Handbook of Parvoviruses, ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). The ITR sequences are about 145 bp in length. Preferably, substantially the entire ITR coding sequences are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the art. (See, e.g., Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520-532 (1996)). An example of such a molecule useful in the present invention is a cis-acting plasmid containing a transgene in which the selected transgene sequence and associated regulatory elements are flanked by AAV 5' and 3' ITR sequences. In one embodiment, the ITRs are from an AAV different from the one that provides the capsid. In one embodiment, the ITR sequences are from AAV2. A shortened version of the 5' ITR, called AITR, has been described in which the D sequence and the terminal separation site (trs) have been removed. In other embodiments, the full-length AAV 5' and 3' ITRs are used. However, ITRs from other AAV sources may be selected. If the ITR source is from AAV2 and the AAV capsid is from a different AAV source, the resulting vector may be called pseudotyped. However, other configurations of these elements may be suitable.

In addition to the essential elements specified above for the recombinant AAV vector, the vector also includes the necessary conventional control elements that are operably linked to the transgene in a manner that allows for its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus obtained in the present invention. As used herein, operably linked sequences include both expression control sequences that are adjacent to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest.

Regulatory control elements typically comprise a promoter sequence as part of the expression control sequences, for example located between the selected 5' ITR sequence and the coding sequence. Constitutive promoters, regulated promoters [see, for example, WO 2011/126808 and WO 2013/04943], tissue-specific promoters or a promoter responsive to physiological signals can be used in the vectors described herein. The promoter(s) may be selected from a variety of sources, such as the cytomegalovirus (CMV) immediate early enhancer/promoter, the SV40 early enhancer/promoter, the JC polymovirus promoter, the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters, the herpes simplex virus (HSV-1) latency-associated promoter (LAP), the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, the neuron-specific promoter (NSE), the platelet-derived growth factor (PDGF) promoter, hSYN, the melanin-concentrating hormone (MCH) promoter, CBA, the matrix metalloprotein (MPP) promoter, and the chicken beta-actin promoter. In addition to the promoter, the vector may contain one or more other

- 19 048431 other suitable transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA, such as WPRE; sequences that enhance translation efficiency (i.e., a Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. An example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include those that are appropriate for the desired characteristics of the target tissue. In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette comprises two or more expression enhancers. These enhancers may be the same or may be different from each other. For example, the enhancer may comprise a CMV immediate early enhancer. This enhancer may be present in two copies that are adjacent to each other. Alternatively, the duplicated copies of the enhancer may be separated by one or more sequences. In another embodiment, the expression cassette further comprises an intron, such as a chicken beta-actin intron. Other suitable introns include those known in the art, such as those described in WO 2011/126808. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA.

Optionally, one or more sequences can be selected to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence, which can be designed upstream of the polyA sequence and downstream of the coding sequence [see, e.g., M. A. Zanta-Boussif et al., Gene Therapy (2009) 16: 605–619.

These rAAVs are particularly suitable for gene delivery for therapeutic purposes, as well as for immunization, including the induction of protective immunity. In addition, the compositions of the invention can also be used to produce a desired gene product in vitro. For in vitro production, the desired product (e.g., a protein) can be obtained from a desired culture after transfecting host cells with an rAAV containing a molecule encoding the desired product and culturing the cell culture under conditions that allow expression. The expressed product can, if desired, then be purified and isolated. Suitable methods for transfection, cell culturing, purification, and isolation are known to those skilled in the art.

In certain embodiments, the rAAV or composition as provided herein does not comprise an influenza antibody or immunoglobulin construct. In certain embodiments, the rAAV or composition as provided herein does not comprise a sequence encoding SMN.

Therapeutic genes and gene products

Suitable products encoded by a transgene include a variety of gene products that replace a defective or deficient gene, inactivate or knock out, or knock down, or reduce the expression of a gene that is expressed at an undesirably high level, or deliver a gene product that has a desired therapeutic effect. In most embodiments, the therapy will be somatic gene therapy, i.e., gene transfer into a cell of the body that does not produce sperm or eggs. In certain embodiments, the proteins expressed by the transgenes have the sequence of native human sequences. However, in other embodiments, synthetic proteins are expressed. Such proteins may be intended for the treatment of humans or, in other embodiments, intended for the treatment of animals, including domestic animals, such as canine or feline populations, or for the treatment of livestock or other animals that come into contact with human populations.

Examples of suitable gene products may include those associated with familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, and rare or orphan diseases. Examples of such a rare disease may include, among others, spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome (e.g., with methyl-CpG-binding protein 2 (MeCP2); UniProtKB - P51608), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Friedrichs ataxia (e.g., frataxin), progranulin (PRGN) (associated with non-Alzheimer's brain degeneration, including frontotemporal dementia (FTD), progressive nonfluent aphasia (PNFA), and semantic dementia). See, for example, www.orpha.net/consor/cgi-bin/Disease_Search_List.php;rarediseases.info.nih.gov/diseases.

Examples of suitable genes may include, for example, hormones and growth and differentiation factors, including, but not limited to, insulin, glucagon, glucagon-like peptide-1 (GLP-1), growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO) (including, for example, human,

- 20 048431 canine or feline EPO), connective tissue growth factor (CTGF), neutrophil factors including, for example, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (cFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-1 and IGF-II), any of the transforming growth factor-α superfamily including TGFa, activins, inhibins, or any of the bone morphogenetic proteins (BMPs) BMP 1-15, any of the heregulin/neuregulin/BCC/neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, any of the semaphorin/collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog, and tyrosine hydroxylase.

Other suitable transgene products include proteins that regulate the immune system, including, but not limited to, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-1 to IL-36 (including, for example, human interleukins IL-1, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-8, IL-12, IL-11, IL-12, IL-13, IL-18, IL-31, IL-35), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte macrophage colony stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β and γ, stem cell factor, ligand flk-2/flt3. Gene products produced by the immune system may also be useful in the present invention. These include, but are not limited to, IgG, IgM, IgA, IgD, and IgE immunoglobulins, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. For example, in certain embodiments, rAAV antibodies may be designed to deliver canine or feline antibodies, such as, for example, anti-IgE, anti-IL31, anti-CD20, anti-NGF, anti-GnRH. Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2, CD59, and C1 esterase inhibitor (C1-INH).

However, other suitable gene products include any of the receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins and proteins of the immune system. The invention encompasses receptors for cholesterol regulation and/or lipid modulation, including the low-density lipoprotein receptor (LDL), the high-density lipoprotein receptor (HDL), the very-low-density lipoprotein receptor (VLDL) and the phagocytic receptor. The invention also encompasses gene products such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors and estrogen receptors, vitamin D receptors and other nuclear receptors. In addition, suitable products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT box binding proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS binding protein, STAT, GATA box binding proteins such as GATA-3, and the Forkhead family of winged helix proteins.

Other useful gene products include carbamoyl synthetase I, ornithine transcarbamylase (OTC), arginosuccinate synthetase, arginosuccinate lyase (ASL) for treatment of arginosuccinate lyase deficiency, arginase, fumaryl acetate hydrolase, phenylalanine hydroxylase, alpha-1-antitrypsin, Rhesus alpha-fetoprotein (AFP), Rhesus human chorionic gonadotropin (hCG), glucose-6-phosphatase, porphobilinogen deaminase, cystathionine beta synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H protein, T protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and the dystrophin gene product [e.g., mini- or micro-dystrophin]. However, other useful gene products include enzymes such as those that may be useful in enzyme replacement therapy, which is beneficial in a variety of conditions resulting from deficient enzyme activity. For example, enzymes that contain mannose-6-phosphate may be used in therapies against lysosomal storage diseases (e.g., a useful gene includes that encoding β-glucuronidase (GUSB)).

In certain embodiments of the invention, rAAV can be used in gene editing systems, which system can include a single rAAV or co-administration of several parent rAAVs. For example, rAAV can be engineered to deliver SpCas9, SaCas9, ARCUS, Cpf1 and other suitable gene editing constructs.

However, other useful gene products include those used to treat hemophilia, including hemophilia B (including factor IX) and hemophilia A (including factor VIII and its variants such as the light chain and heavy chain heterodimer and the B-deleted domain; U.S. Patent No. 6,200,560 and U.S. Patent No. 6,221,349). In some embodiments, the minigene contains

- 21 048 431 contains the first 57 base pairs of the heavy chain of factor VIII, which encodes a 10 amino acid signal sequence, as well as a human growth hormone (hGH) polyadenylation sequence. In alternative embodiments of the invention, the minigene further comprises domains A1 and A2, as well as 5 amino acids from the N-terminus of domain B and/or 85 amino acids from the C-terminus of domain B, as well as domains A3, C1 and C2. In still other embodiments of the invention, nucleic acids encoding the heavy chain and light chain of factor VIII are provided in a single minigene, separated by 42 nucleic acids encoding 14 amino acids of domain B [U.S. Patent No. 6,200,560].

Other useful gene products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides, which include a non-naturally occurring amino acid sequence containing amino acid insertions, deletions, or substitutions. For example, single-chain engineered immunoglobulins may be useful for some immunocompromised patients. Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which can be used to reduce target overexpression.

Reduction and/or modulation of gene expression is particularly desirable for the treatment of hyperproliferative conditions characterized by hyperproliferating cells, such as cancer and psoriasis. Target polypeptides include those polypeptides that are produced exclusively or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include polypeptides encoded by oncogenes such as myb, myc, fyn, and the bcr/abl translocation gene, ras, src, P53, neu, trk, and EGRF. In addition to oncogenic products as target antigens, target polypeptides for anticancer treatment and protective regimens include variable regions of antibodies made by B-cell lymphocytes and variable regions of T-cell receptors of T-cell lymphocytes, which, in some embodiments of the invention, are also used as target antigens for an autoimmune disease. Other tumor-associated polypeptides can be used as target polypeptides, such as polypeptides that are found at higher levels in tumor cells, including the polypeptide recognized by monoclonal antibody 171A and folate-binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those that may be useful in treating individuals suffering from autoimmune diseases and disorders by eliciting a broad protective immune response against targets associated with autoimmunity, including cellular receptors and cells that produce self-directed antibodies. T-cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis. Each of these diseases is characterized by T cell receptors (TCRs) that bind to endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases.

Also exemplary genes that can be delivered via rAAV include, but are not limited to, glucose-6-phosphatase associated with glycogen storage disease or deficiency type 1A (GSD1), phosphoenolpyruvate carboxykinase (PEPCK) associated with PEPCK deficiency; cyclin-dependent kinase-like protein 5 (CDKL5), also known as serine/threonine kinase 9 (STK9), associated with seizures and severe neurodevelopmental disorder; galactose-1-phosphate uridyl transferase associated with galactosemia; phenylalanine hydroxylase associated with phenylketonuria (PKU); branched-chain alpha-keto acid dehydrogenase associated with maple syrup urine disease; fumaryl acetoacetate hydrolase associated with tyrosinemia type 1; Methylmalonyl-CoA mutase associated with methylmalonic acidemia; Medium-chain fatty acid acyl-CoA dehydrogenase associated with medium-chain fatty acid acetyl-CoA deficiency; Ornithine transcarbamylase (OTC) associated with ornithine transcarbamylase deficiency; Argininosuccinate synthetase (ASS1) associated with citrullinemia; Lecithin-cholesterol acyltransferase deficiency (LCAT); Methylmalonic acidemia (MMA); Niemann-Pick disease, type C1; Propionibacterium adenosine (PA); Low-density lipoprotein receptor protein (LDLR) associated with familial hypercholesterolemia (FH); UDPglucuronosyltransferase associated with Crigler-Najjar disease; adenosine deaminase associated with severe combined immunodeficiency disease; hypoxanthine-guanine phosphoribosyl transferase associated with gout and Lesch-Nyan syndrome; biothymidase associated with biothymidase deficiency; alpha-galactosidase A (a-Gal A) associated with Fabry disease; ATP7B associated with Wilson's disease; beta-glucocerebrosidase associated with Gaucher disease types 2 and 3; peroxisomal membrane protein 70 kDa associated with Zellweger syndrome; arylsulfatase A (ARSA) associated with metachromatic leukodystrophy, galactocerebrosiderase enzyme (GALC) associated with Krabbe disease, alpha-glucosidase (GAA) associated with Pompe disease; sphingomyelinase gene (SMPD1) associated with Niemann Pick disease type A; argininosuccinate synthase associated with adult-onset citrullinemia type II (CTLN2); carbamoyl phosphate synthase 1 (CPS1), associated with urea cycle disorders; survival motor neuron (SMN) protein associated with spinal cord injury

- 22 048431 neck atrophy; ceramidase associated with Farbe lipogranulomatosis; b-hexosaminidase associated with GM2-raHrauo3ugo3OM and Tay-Sachs and Sanhoff diseases; aspartylglucosaminidase associated with aspartylglucosaminuria; a-fucosidase associated with fucosidosis; α-mannosidase associated with alpha-mannosidosis; porphobilinogen deaminase associated with acute intermittent porphyria (AIP); alpha-1-antitrypsin for the treatment of alpha-1 antitrypsin deficiency (emphysema); erythropoietin for the treatment of anemia due to thalassemia or renal failure; vascular endothelial growth factor, angiopoietin-1 and fibroblast growth factor for the treatment of ischemic diseases; thrombomodulin and a tissue factor pathway inhibitor for the treatment of occlusion of blood vessels, as provided, for example, in atherosclerosis, thrombosis or embolism; aromatic amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; beta-adrenergic receptors, anti-sense or mutant form of phospholamban, sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2) and cardiac adenylyl cyclase for the treatment of congestive heart failure; a tumor suppressor gene such as p53 for the treatment of various types of cancer; a cytokine such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancers; dystrophies or minidystrophin, and utrophin or miniutrophin for the treatment of muscular dystrophies; and insulin or GLP-1, for the treatment of diabetes mellitus.

Additional genes and diseases of interest include, for example, dystonin gene-related diseases such as hereditary sensory and autonomic neuropathies type VI (the DST gene encodes dystonin; dual AAV vectors may be required due to the size of the protein (~7570 aa); SCN9A-related diseases in which loss of function mutants cause inability to sense pain and gain of function mutants cause pain conditions such as erythromelalgia. Another condition is Charcot-Marie-Tooth syndrome types 1F and 2E due to mutations in the NeFL (neurofilament light chain) gene, characterized by progressive peripheral motor and sensory neuropathy with variable clinical and electrophysiological expression.

In certain embodiments, the rAAV described herein may be used in the treatment of mucopolysaccharidosis (MPS) disorders. Such rAAV may comprise a transferable nucleic acid sequence encoding a-L-iduronidase (IDUA) for the treatment of MPS I (Hurler, Hurler-Shyat, and Shyat syndromes); a nucleic acid sequence encoding iduronate-2-sulfatase (IDS) for the treatment of MPS II (Hunter syndrome); a nucleic acid sequence encoding sulfamidase (SGSH) for the treatment of MPSIII A, B, C, and D (Sanfilippo syndrome); a nucleic acid sequence encoding N-acetylgalactosamine-6-sulfate sulfatase (GALNS) for the treatment of MPS IV A and B (Morquio syndrome); a nucleic acid sequence encoding arylsulfatase B (ARSB) for the treatment of MPS VI (Maroteaux-Lamy syndrome); a nucleic acid sequence encoding hyaluronidase for the treatment of MPSI IX (hyaluronidase deficiency), and a nucleic acid sequence encoding beta-glucuronidase for the treatment of MPS VII (Sly syndrome).

Immunogenic transgenes

In some embodiments, an rAAV vector comprising a nucleic acid encoding a gene product associated with cancer (e.g., tumor suppressors) can be used to treat cancer by administering rAAV carrying the rAAV vector to a subject having cancer. In some embodiments, an rAAV vector comprising a nucleic acid encoding a small interfering nucleic acid (e.g., shRNA, microRNA) that inhibits the expression of a gene product associated with cancer (e.g., oncogenes) can be used to treat cancer by administering rAAV carrying the rAAV vector to a subject having cancer. In some embodiments, an rAAV vector comprising a nucleic acid encoding a gene product associated with cancer (or a functional RNA that inhibits the expression of a gene associated with cancer) can be used for research purposes, such as to study cancer or to identify therapeutic agents that treat cancer. The following is a non-limiting list of illustrative genes known to be associated with cancer development (e.g., oncogenes and tumor suppressors):

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AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AKI, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2, BHLHB2, BLMH, BRAF, BRCA1, BRCA2, VTK, CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, C0L1A1, COL6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1, CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8, DLG3, DVL1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2, ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES, FGFR1, FGR, FKBP8, FN1, FOS, F0SL1, F0SL2, FOXG1A, FOXO1A, FRAP1, FRZB, FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2, GNB2L1, GPR39, GRB2, GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HMMR, HPRT1, HRB, HSPA4, HSPA5, HSPA8, HSPB1, HSPH1, HYAL1, HYOU1, ICAM1, ID1, ID2, IDUA, IER3, IFITM1, IGF1R, IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1, IRF3, ITGA3, ITGA6, ITGB4, JAKI, JARID1A, JUN, JUNB, JUND, K-ALPHA-1, KIT, KITLG, KLK10, KPNA2, KRAS2, KRT18, KRT2A, KRT9, LAMB1, LAMP2, LCK, LCN2, LEP, LITAF, LRPAP1, LTF, LYN, LZTR1, MADH1, MAP2K2, MAP3K8, MAPK12, MAPK13, MAPKAPK3, MAPRE1, MARS, MASI, MCC, MCM2, MCM4, MDM2, MDM4, MET, MGST1, MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA, MSH2, MSH6, MT3, MYB, MYBL1, MYBL2, MYC, MYCL1, MYCN, MYD88, MYL9, MYLK, ΝΕΟΙ, NF1, NF2, NFKB1, NFKB2, NFSF7, NID, NINE, NMBR, NME1, NME2, NME3, NOTCH 1, NOTCH2, NOTCH4, NPM1, NQO1, NR1D1, NR2F1, NR2F6, NRAS, NRG1, NSEP1, OSM, PA2G4, PABPC1, PCNA, PCTK1, PCTK2, PCTK3, PDGFA, PDGFB, PDGFRA, PDPK1, PEA15, PFDN4, PFDN5, PGAM1, PHB, PIK3CA, PIK3CB, PIK3CG, PIM1, PKM2, PKMYT1, PLK2, PPARD, PPARG, PPIH, PPP1CA, PPP2R5A, PRDX2, PRDX4, PRKAR1A, PRKCBP1, PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA, PTN, PTPRN, RAB5A, RAC1, RAD50, RAFI, RALBP1, RAP1A, RARA, RARB, RASGRF1, RBI, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB, RHOC, RHOD, RIPK1, RPN2, RPS6 KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D, SEPPI, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH , SIAH2, SIVA, SIVA TP53, SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, sphingomyelin phosphodiesterase 1 (SMPD1), SNAI2, SND1, SNRPB2, S0CS1, S0CS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STAT2, STAT3, STAT5B , STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, TK1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TNK1, TOBI, TP53, TP53BP2, TP5313, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL, VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB, YWHAZ, ZAP70 and ZNF9.

The rAAV vector may comprise as a transgene a nucleic acid encoding a protein or functional RNA that modulates apoptosis. The following is a non-limiting list of genes associated with apoptosis and nucleic acids encoding the products of these genes and their homologues and encoding small interfering nucleic acids (e.g., shRNA, microRNA) that inhibit the expression of these genes and their homologues are useful as transgenes in certain embodiments of the invention:

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RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3, BAG4, BAKI, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2, BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L, BOK, BRAF, CARDIO, CARD11, NLRC4, CARD14, NOD2, NODI, CARD6, CARDS, CARDS, CASP1, CASP10, CASP14, CASP2 CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, GADD45A, GDNF, HRK, IGF1R, LTA, LTBR, MCL1, NOL3, PYCARD, RIPK1, RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRSF12A, TNFRSF14, TNFRSF19, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25, CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18, CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4 and TRAF5.

Useful gene products also include microRNAs. MicroRNAs and other small interfering nucleic acids that regulate gene expression by cleavage/degradation of a target RNA transcript or translational repression of a target messenger RNA (mRNA). MicroRNAs are typically initially expressed as completed 19-25 untranslated RNA products. MicroRNAs exert their activity through sequence-specific interactions with the 3' untranslated regions (UTRs) of target mRNAs. These endogenously expressed microRNAs form hairpin-shaped precursors that are subsequently processed into duplex microRNA and then into a mature single-stranded microRNA molecule. This mature microRNA directs a multiprotein complex, miRISC, which identifies the target site, such as the 3' UTRs, of target mRNAs based on their complementarity to the mature microRNA.

The following non-limiting list of microRNA genes and their homologues are useful as genes or as targets for small interfering nucleic acids encoded by genes (e.g., microRNA sponges, antisense oligonucleotides, TuD-RNA) in certain embodiments of the methods:

hsalet-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c, hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*, hsa-let-7f, hsa-let-7f-l*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*, hsa-let-71, hsa-let-71*, hsa-miR-1, hsa-miR-100, hsa-miR-100*, hsa-miR-101 , hsa-miR101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*, hsa-miR-106a, hsa-miR-106a*, hsa-miR106b, hsa-miR-106b*, hsa-miR-107, hsa-miR-lOa, hsa-miR-10a*, hsa-miR-lOb, hsa-miR10b*, hsa- miR-1178, hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsamiR-1183, hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR- 1201, hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206, hsa-miR1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122, hsa-miR-122*, hsa-miR-12243p , hsa-miR-1224-5p, hsa-miR-1225-Зр, hsa-miR-1225-5p, hsa-miR-1226, hsa-miR1226*, hsa-miR-1227, hsa-miR-1228, hsa-miR-1228*, hsa-miR- 1229, hsa-miR-1231, hsamiR-1233, hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124, hsamiR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246, hsa-miR -1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251, hsa-miR-1252, hsa-miR1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b, hsa-miR- 1256, hsa-miR-1257, hsamiR-1258, hsa-miR-1259, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b, hsa-miR125b-l*, hsa-miR-125b-2*, hsa-miR-126, hsa-miR-126* , hsa-miR-1260, hsa-miR-1261, hsa-miR-1262, hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR1267, hsa-miR-1268, hsa-miR-1269, hsa-miR- 1270, hsa-miR-1271, hsa-miR-1272, hsamiR-1273, hsa-miR-127-Зр, hsa-miR-1274a, hsa-miR-1274b, hsa-miR-1275, hsa-miR-127- 25 048431

5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278, hsa-miR-1279, hsa-miR-128, hsa-miR1280, hsa-miR-1281, hsa-miR-1282, hsa- miR-1283, hsa-miR-1284, hsa-miR-1285, hsamiR-1286, hsa-miR-1287, hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291, hsa-miR -1292, hsa-miR-1293, hsa-miR-129-Zr, hsa-miR-1294, hsa-miR1295, hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299, hsamiR-1300, hsa-miR- 1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304, hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a, hsa-miR-130a*, hsa -miR130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*, hsa-miR-1321, hsa-miR-1322, hsamiR-1323, hsa-miR-1324, hsa-miR-133a, hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsamiR-135a* , hsa-miR-135b, hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-137, hsa-miR-138, hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p, hsamiR-140-Zr, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*, hsa-miR-142-Зр, hsa-miR142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144, hsa-miR- 144*, hsa-miR-145, hsa-miR145*, hsa-miR-146a, hsa-miR-146a*, hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b, hsa-miR-148a, hsa-miR -148a*, hsa-miR-148b, hsa-miR-148b*, hsa-miR149, hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-Зр, hsa-miR-151-5p, hsamiR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154 *, hsa-miR-155, hsa-miR-155*, hsamiR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*, hsa-miR-16, hsa-miR-16-1*, hsamiR-16-2*, hsa-miR-17, hsa- miR-17*, hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b, hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*, hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*, hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*, hsa-miR-187, hsa-miR-187 *, hsa-miR-188-Зр, hsa-miR-188-5p, hsa-miR-18a, hsa-miR-18a*, hsa-miR-18b, hsa-miR18b*, hsa-miR-190, hsa-miR-190b, hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR192*, hsa-miR -193a-3p, hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194*, hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a *, hsa-miR-196b, hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsamiR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR-19b-l*, hsa-miR-19b-2* , hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b, hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR202, hsa-miR-202*, hsa-miR-203, hsa-miR-204, hsa -miR-205, hsa-miR-206, hsa-miR208a, hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*, hsa-miR21, hsa-miR-21*, hsa-miR-210, hsa-miR-211, hsa-miR-212, hsa -miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b, hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*, hsa-miR-219-l- 3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22, hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221, hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*, hsa-miR-224, hsa-miR-23a, hsa-miR-23a*, hsa-miR-23b, hsa-miR-23b*, hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*, hsa-miR-26a, hsa-miR-26a-l*, hsa-miR-26a-2*, hsa-miR-26b, hsa-miR-26b*, hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-Зр, hsa-miR-28-5p, hsa -miR-296-Зр, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298, hsa-miR

- 26 048431

299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b, hsa-miR-296-1*, hsa-miR-296-2*, hsa -miR-29c, hsa-miR-29c*, hsa-miR-300, hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b, hsa-miR-302b*, hsa-miR-302c, hsa-miR302c*, hsa -miR-302d, hsa-miR-302d*, hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsamiR-30a*, hsa-miR-30b, hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-l*, hsa-miR-30c -2*, hsa-miR-30d, hsa-miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hsa-miR-31*, hsamiR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa -miR-320c, hsa-miR-320d, hsamiR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p, hsa-miR-324-5p, hsa-miR-325, hsa-miR326, hsa-miR-328, hsa-miR-329, hsa-miR-33O-3p, hsa-miR- 330-5p, hsa-miR-331-3p, hsamiR-331-5p, hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p, hsa-miR338-3p, hsa-miR-338-5p, hsa-miR-339-3p , hsa-miR-339-5p, hsa-miR-33a, hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340, hsa-miR-340*, hsa-miR-342-3p, hsa-miR-3425p, hsa-miR-345, hsa-miR-346, hsa-miR- 34a, hsa-miR-34a*, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p, hsa-miR-34c-5p, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p, hsa-miR -362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367, hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370, hsa-miR-371-3p, hsa-miR-371-5p, hsamiR-372, hsa -miR-373, hsa-miR-373*, hsa-miR-374a, hsa-miR-374a*, hsa-miR-374b, hsamiR-374b*, hsa-miR-375, hsa-miR-376a, hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa -miR-377, hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*, hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-384, hsamiR-409-Зр, hsa-miR^409-5p, hsa-miR-410, hsa-miR-411, hsa-miR-411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR -424, hsa-miR424*, hsa-miR-425, hsa-miR-425*, hsa-miR^429, hsa-miR-431, hsa-miR-431*, hsa-miR432, hsa-miR-432*, hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa- miR450a, hsa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR- 455-Zr, hsa-miR-455-5p, hsa-miR483-3p, hsa-miR-483-5p, hsa-miR-484, hsa-miR-485-Зр, hsa-miR-485-5p, hsa-miR-4863p, hsa-miR-486-5p, hsa-miR-487a, hsa-miR-487b, hsa-miR-488, hsa- miR-488*, hsa-miR489, hsa-miR-490-Зр, hsa-miR-490-5p, hsa-miR-491-Зр, hsa-miR-491-5p, hsa-miR-492, hsa-miR-493, hsa-miR^493*, hsa-miR-494, hsa -miR-495, hsa-miR-496, hsa-miR-497, hsamiR-497*, hsa-miR-498, hsa-miR-499-Зр, hsa-miR-499-5p, hsa-miR-500, hsa-miR-500*, hsa-miR-501-Зр, hsa-miR-501-5p, hsa-miR-502 -Zr, hsa-miR-502-5p, hsa-miR-503, hsamiR-504, hsa-miR-505, hsa-miR-505*, hsa-miR-506, hsa-miR-507, hsa-miR-508-Зр, hsamiR-508-5p, hsa-miR-509-3 -5p, hsa-miR-509-Зр, hsa-miR-509-5p, hsa-miR-510, hsamiR-511, hsa-miR-512-Зр, hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hsamiR-513b, hsa-miR-513c, hsa-miR-514, hsa- miR-515-Зр, hsa-miR-515-5p, hsa-miR-516a3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*, hsa-miR-517a, hsa-miR-517b, hsamiR-517c, hsa-miR-518a-3p , hsa-miR-518a-5p, hsa-miR-518b, hsa-miR-518c, hsa-miR518c*, hsa-miR-518d-3p, hsa-miR-518d-5p, hsa-miR-518e, hsa-miR-518e*, hsa-miR-518f, hsa-miR-518f*, hsa-miR -519a, hsa-miR-519b-3p, hsa-miR-519c-3p, hsa-miR-519d, hsa

- 27 048431 miR-519e, hsa-miR-519e*, hsa-miR-520a-3p, hsa-miR-520a-5p, hsa-miR-520b, hsa-miR520c-3p, hsa-miR-520d-3p, hsa-miR-520d-5p, hsa-miR-520e, hsa-miR-520f, hsa-miR520g, hsa-miR-520h, hsa-miR-521, hsa-miR-522, hsa-miR-523, hsa-miR-524-Зр, hsa- miR524-5p, hsa-miR-525-Зр, hsa-miR-525-5p, hsa-miR-526b, hsa-miR-526b*, hsa-miR-5323p, hsa-miR-532-5p, hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR- 542-Zr, hsamiR-542-5p, hsa-miR-543, hsa-miR-544, hsa-miR-545, hsa-miR-545*, hsa-miR-548a-3p, hsa-miR-548a-5p, hsa-miR-548b-3p, hsa-miR-5486-5p , hsa-miR-548c-3p, hsa-miR-548c5p, hsa-miR-548d-3p, hsa-miR-548d-5p, hsa-miR-548e, hsa-miR-548f, hsa-miR-548g, hsamiR-548h, hsa-miR-548i, hsa-miR-548j, hsa-miR-548k, hsa- miR-5481, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o, hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a, hsa-miR-551b, hsa -miR-551b*, hsa-miR-552, hsa-miR-553, hsa-miR-554, hsa-miR-555, hsa-miR-556-Зр, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558, hsa-miR559, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564, hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570, hsa-miR-571, hsa-miR-572, hsa-miR-573, hsamiR-574-Зр, hsa- miR-574-5p, hsa-miR-575, hsa-miR-576-Зр, hsa-miR-576-5p, hsa-miR577, hsa-miR-578, hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582-Зр, hsa-miR582-5p, hsa-miR-583, hsa- miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587, hsa-miR588, hsa-miR-589, hsa-miR-589*, hsa-miR-590-Зр, hsa-miR-590-5p, hsa-miR-591, hsamiR-592, hsa -miR-593, hsa-miR-593*, hsa-miR-595, hsa-miR-596, hsa-miR-597, hsamiR-598, hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR604, hsa-miR-605, hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610, hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-Зр, hsa-miR-6155p, hsa-miR-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618, hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623, hsa-miR-624, hsa-miR-624*, hsa-miR-625, hsamiR -625*, hsa-miR-626, hsa-miR-627, hsa-miR-628-Зр, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*, hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR -633, hsa-miR-634, hsamiR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639, hsa-miR-640, hsa-miR641, hsa-miR-642, hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR- 646, hsa-miR-647, hsa-miR-648, hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-653, hsamiR-654-Зр, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-663b, hsamiR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668, hsa-miR -671-Зр, hsa-miR-671-5p, hsa-miR-675, hsa-miR-7, hsa-miR-708, hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*, hsamiR-720, hsa-miR-744, hsa-miR -744*, hsa-miR-758, hsa-miR-760, hsa-miR-765, hsamiR-766, hsa-miR-767-Зр, hsa-miR-767-5p, hsa-miR-768-Зр, hsa-miR-768-5p, hsa-miR769-3p, hsa-miR-769-5p, hsa-miR- 770-5p, hsa-miR-802, hsa-miR-873, hsa-miR-874, hsamiR-875-Zr, hsa-miR-875-5p, hsa-miR-876-Zr, hsa-miR-876-5p, hsa-miR-877, hsa-miR877*, hsa-miR -885-Zr, hsa-miR-885-5p, hsa-miR-886-Зр, hsa-miR-886-5p, hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889, hsa-miR-890, hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa -miR-892b, hsa-miR-9, hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsamiR-922, hsa-miR-923, hsa-miR-924, hsa-miR-92a, hsa-miR-92a-l*, hsa-miR-92a-2 *, hsa-miR-92b, hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933, hsa-miR-934, hsamiR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-miR-939, hsa-miR-940, hsa-miR941, hsa-miR-942, hsa-miR-943, hsa-miR-944, hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98, hsa-miR-99a, hsa-miR-99a*, hsa-miR-99b and hsa-miR-99b*.

For example, a microRNA targeting chromosome 8 open reading frame 72 (C9orf72), which expresses superoxide dismutase (SOD1), which is associated with amyotrophic lateral sclerosis (ALS), may be of interest.

MicroRNA inhibits the function of the mRNAs it targets and, as a result, inhibits

- 28 048431 expression of polypeptides encoded by these mRNAs. Thus, blocking (partially or completely) the activity of a microRNA (e.g., silencing a microRNA) can effectively induce or restore the expression of a polypeptide whose expression is inhibited (derepression of the polypeptide). In one embodiment of the invention, derepression of polypeptides encoded by mRNAs to which the microRNA is targeted is accomplished by inhibiting the activity of the microRNA in cells by any of a variety of methods. For example, blocking the activity of a microRNA can be achieved by hybridization with a small interfering nucleic acid (e.g., an antisense oligonucleotide, a microRNA sponge, TuD-RNA) that is complementary or substantially complementary to the microRNA, thereby blocking the interaction of the microRNA with its target mRNA. As used herein, a small interfering nucleic acid that is substantially complementary to a microRNA is one that is capable of hybridizing to a microRNA and blocking the activity of the microRNA. In some embodiments, a small interfering nucleic acid that is substantially complementary to a microRNA is a small interfering nucleic acid that is complementary to a microRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases. A microRNA inhibitor is an agent that blocks the function, expression, and/or processing of a microRNA. For example, these molecules include, but are not limited to, specific antisense miRNAs, miRNA sponges, hard decoy RNAs (TuD-RNAs), and miRNA oligonucleotides (double-stranded, hairpin, short oligonucleotides) that inhibit the interaction of miRNAs with the Drosha complex.

In addition, other useful genes may include genes encoding immunoglobulins that confer passive immunity to a pathogen. An immunoglobulin molecule is a protein comprising the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently linked together and capable of specifically combining with an antigen. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The terms antibody and immunoglobulin may be used interchangeably herein.

An immunoglobulin heavy chain is a polypeptide that comprises at least a portion of an immunoglobulin antigen-binding domain and at least a portion of an immunoglobulin heavy chain variable region or at least a portion of an immunoglobulin heavy chain constant region. Thus, an immunoglobulin-derived heavy chain has significant regions of amino acid sequence homology with a member of the immunoglobulin gene superfamily. For example, the heavy chain in a Fab fragment is an immunoglobulin-derived heavy chain. An immunoglobulin light chain is a polypeptide that comprises at least a portion of an immunoglobulin antigen-binding domain and at least a portion of an immunoglobulin light chain variable region or at least a portion of a constant region. Thus, an immunoglobulin-derived light chain has significant regions of amino acid sequence homology with a member of the immunoglobulin gene superfamily. An immunoadhesin is a chimeric antibody-like molecule that combines the functional domain of a binding protein, typically a receptor, ligand, or cell adhesion molecule, with the constant domains of an immunoglobulin, typically including the hinge and Fc regions.

The antigen-binding fragment (Fab) is the region on an antibody that binds to antigens. It consists of one constant and one variable region from each heavy and light chain.

The design of the antipathogen is selected based on the disease-causing agent (pathogen) against which protection is sought. These pathogens may be of viral, bacterial or fungal origin, and may be used to prevent infection in humans, against human disease, or in non-human mammals or other animals to prevent veterinary disease.

rAAV may include genes encoding antibodies, and particularly neutralizing antibodies, against a viral pathogen. Such antiviral antibodies may include influenza antibodies directed against one or more of influenza A, influenza B, and influenza C. Influenza A viruses are the most virulent human pathogens. Influenza A serotypes that have been associated with pandemics include H1N1, which caused the Spanish flu in 1918 and swine flu in 2009; H2N2, which caused the Asian flu in 1957; H3N2, which caused the Hong Kong flu in 1968; H5N1, which caused the avian flu in 2004; H7N7; H1N2; H9N2; H7N2; H7N3; and H10N7. Other target pathogenic viruses include arenaviruses (including Funin, Machupo, and Lassa), filoviruses (including Marburg and Ebola), hantaviruses, picornaviruses (including rhinoviruses, echovirus), coronaviruses, paramyxoviruses, morbillivirus, respiratory syncytial virus, togavirus, coxsackievirus, JC virus, parvovirus B19, parainfluenza, adenoviruses, reoviruses, smallpox (Variola major (smallpox)) and Vaccinia (cowpox) of the poxvirus family, and varicella (pseudorabies). Viral hemorrhagic fevers are caused by members of the arenavirus (Lassa fever) family (this family is also associated with lymphocytic choriomeningitis (LCM)), filovirus (Ebola virus), and hantavirus (Puumala). Members

- 29 048431 picornaviruses (subfamily Rhinovirus) are associated with the common cold in humans. A family of coronaviruses that includes a number of non-human viruses such as infectious bronchitis virus (birds), porcine transmissible gastrointestinal virus (pig), porcine hemagglutinatin encephalomyelitis virus (pig), feline infectious peritonitis virus (cat), feline enteric coronavirus (cat), canine coronavirus (dog). Human respiratory coronaviruses that have been putatively associated with the common cold, hepatitis non-A, B, or C, and sudden acute respiratory syndrome (SARS). The paramyxovirus family includes parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, rubulavirus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, morbillivirus, which includes measles and canine distemper, and pneumovirus, which includes respiratory syncytial virus (RSV). The parvovirus family includes feline parvovirus (feline enteritis), feline panleukopeniavirus, canine parvovirus, and porcine parvovirus. The adenovirus family includes viruses (EX, AD7, ARD, O.B.) that cause respiratory disease. Thus, in certain embodiments of the invention, an rAAV vector as described herein can be engineered to express an anti-Ebola virus antibody, such as 2G4, 4G7, 13C6, an anti-influenza antibody, such as FI6, CR8033, and an anti-RSV antibody, such as palivizumab, motavizumab.

A neutralizing antibody construct against bacterial pathogens may also be selected for use in the present invention. In one embodiment, the neutralizing antibody construct is directed against the bacteria themselves. In another embodiment, the neutralizing antibody construct is directed against a toxin produced by the bacteria. Examples of airborne bacterial pathogens include, for example, Neisseria meningitidis (meningitis), Klebsiella pneumonia (pneumonia), Pseudomonas aeruginosa (pneumonia), Pseudomonas pseudomallei (pneumonia), Pseudomonas mallei (pneumonia), Acinetobacter (pneumonia), Moraxella catarrhalis, Moraxella lacunata, Alkaligenes, Cardiobacterium, Haemophilus influenzae (flu), Haemophilus parainfluenzae, Bordetella pertussis (whooping cough), Francisella tularensis (pneumonia/fever), Legionella pneumonia (Legionnaires' disease), Chlamydia psittaci (pneumonia), Chlamydia pneumoniae (pneumonia), Mycobacterium tuberculosis (tuberculosis) (TB)), Mycobacterium kansasii (TB), Mycobacterium avium (pneumonia), Nocardia asteroides (pneumonia), Bacillus anthracis (anthrax), Staphylococcus aureus (pneumonia), Streptococcus pyogenes (scarlet fever), Streptococcus pneumoniae (pneumonia), Corynebacteria diphtheria (diphtheria), Mycoplasma pneumoniae (pneumonia).

rAAV may include genes encoding antibodies and, in particular, neutralizing antibodies against a bacterial pathogen such as anthrax, a toxin produced by Bacillius anthracis. Neutralizing antibodies against the protecting agent (ZA), one of three peptides that form the toxoid, have been described. The other two polypeptides consist of the lethal factor (LF) and the edema factor (EF). Anti-ZA neutralizing antibodies have been described as effective in passive immunization against anthrax. See, e.g., U.S. Patent Number 7,442,373; R. Sawada-Hirai et al, J Immune Based Ther Vaccines. 2004; 2: 5. (Online May 12, 2004). Still other neutralizing antibodies against anthrax toxin have been described and/or can be developed. Similarly, neutralizing antibodies against other bacteria and/or bacterial toxins can be used to create AAV-deliverable anti-pathogen constructs as described herein.

Antibodies against infectious diseases may be elicited by parasites or fungi, including, for example, Aspergillus species, Absidia corymbifera, Rhixpus stolonifer, Mucor plumbeaus, Cryptococcus neoformans, Histoplasmcapsulatum, Blastomyces dermatitidis, Coccidioides immitis, Penicillium species, Micropolyspora faeni, Thermoactinomyces vulgaris, Alternaria alternate, Cladosporium species, Helminthosporium species, and Stachybotrys.

rAAV may include genes encoding antibodies and, in particular, neutralizing antibodies against pathogenic factors of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), GBA-Parkinson's disease, rheumatoid arthritis (RA), irritable bowel syndrome (IBS), chronic obstructive pulmonary disease (COPD), cancer, tumors, systemic sclerosis, asthma and other diseases. Such antibodies may be, for example, without limitation, alpha-synuclein, anti-vascular endothelial growth factor (VEGF) antibody (anti-VEGF), anti-VEGFA, anti-PD-1, αntU-PDL1, anti-CTLA-4, anti-TNF-alpha, anti-IL-17, anti-IL-23, anti-IL-21, anti-IL-6, anti-IL-6 receptor antibody, anti-IL-5, anti-IL-7, anti-factor XII antibody, anti-IL-2, anti-HIV, anti-IgE, anti-tumor necrosis factor receptor-1 (TNFR1) antibody, anti-notch 2/3, anti-notch 1, anti-OX40, anti-erb-b2 receptor tyrosine kinase 3 (ErbB3) antibody, anti-ErbB2, anti-beta cell maturation antigen antibody, anti- B-lymphocyte stimulator, anti-CD20, anti-HER2, anti-granulocyte-macrophage colony-stimulating factor, anti-oncostatin M (OSM), anti-lymphocyte activation gene 3 (LAG-3), anti-CCL20, anti-serum amyloid P component (SAP), anti-prolyl hydroxylase inhibitor, anti-CD38, anti-glycoprotein IIb/IIIa, anti-CD52, anti-CD30, anti-IL-1beta, anti-epidermal growth factor receptor, anti-CD25, anti-RANK ligand, anti-C5 protein

- 30 048431 complement system, anti-CDlla, anti _- CD3 receptor antibody, anti-integrin alpha-4 (α4) antibody, anti-RSV F protein antibody and anti-integrin _α4β7 . However, other pathogens and diseases will be obvious to those skilled in the art. Other suitable antibodies may include those useful for the treatment of Alzheimer's disease, such as, for example, anti-beta-amyloid antibody (e.g., crenezumab, solanezumab, aducanumab), anti-beta-amyloid fibril antibodies, anti-beta-amyloid plaque antibodies, anti-tau, bapineuzumab, among others. Other suitable antibodies for treating various indications include those described, for example, in PCT/US2016/058968, filed October 27, 2016, published as WO 2017/075119 A1.

II. Production of rAAV vector.

For use in the production of an AAV viral vector (e.g., recombinant (r) AAV), the expression cassettes can be transferred to any suitable vector, such as a plasmid, which is delivered to a packaging host cell. The plasmids used in the present invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection and packaging methods for host cells are known and/or can be readily developed by one of skill in the art.

Methods for producing and isolating AAVs suitable for use as vectors are known in the art. See, generally, e.g., Grieger & Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin/Biotechnol. 99: 119-145; Buning et al., 2008, Recent developments in adeno-associated virus vector technology, J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. For gene packaging into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassette(s). The cap and rep genes can be placed in trans.

In one embodiment, the expression cassette described herein is inserted into a genetic element (e.g., a shuttle plasmid) that delivers the engineered immunoglobulin sequences carried therein to a host packaging cell for production of a viral vector. In one embodiment, the selected genetic element can be delivered to the AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated beads, viral infection, and protoplast fusion. Stable AAV packaging cells can also be produced. Alternatively, the expression cassettes can be used to create a non-AAV viral vector or to produce antibody mixtures in vitro. Methods used to create such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

The term intermediate AAV or intermediate AAV vector refers to an assembled rAAV capsid that lacks the desired genomic sequences packaged within it. These constructs may also be referred to as an empty capsid. Such a capsid may contain no detectable genomic sequences of the expression cassette, or may contain only partially packaged genomic sequences that are insufficient to achieve expression of the gene product. These empty capsids are nonfunctional for transferring the gene of interest into the host cell.

The recombinant adeno-associated virus (AAV) described herein can be generated using methods known in the art. See, for example, WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7,588,772 B2. Such a method comprises culturing a host cell that comprises a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette consisting of at least AAV inverted terminal repeats (ITRs) and a transgene; and sufficient ancillary functions to allow packaging of the expression cassette into the AAV capsid protein. Methods for producing the capsid, the coding sequences thereof, and methods for producing rAAV viral vectors are described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100(10), 6081-6086 (2003) and US 2013/0045186 A1.

In one embodiment, the invention provides the production of a cell culture suitable for producing recombinant AAVhu68. Such a cell culture comprises a nucleic acid that expresses the AAVhu68 capsid protein in a host cell; a nucleic acid molecule suitable for packaging into the AAVhu68 capsid, such as a vector genome that comprises the AAV ITR and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences that direct expression of that product in the host cell; and sufficient AAV rep functions and adenovirus accessory functions to ensure packaging of the nucleic acid molecule into the recombinant AAVhu68 capsid. In one embodiment, the

- 31 048431 embodiments of the invention, the cell culture consists of mammalian cells (for example, human embryonic kidney 293 cells, among others) or insect cells (for example, baculovirus).

Optionally, the rep functions are provided by an AAV other than hu68. In certain embodiments, at least a portion of the rep functions are taken from AAVhu68. See, for example, the rep sequences encoding the rep proteins of SEQ ID NO: 4 and functional fragments thereof. The AAV rep can be encoded by the nucleic acid sequence of SEQ ID NO: 3. In another embodiment, the rep protein is a heterologous rep protein other than AAVhu68rep, such as, but not limited to, an AAV1 rep protein, an AAV2 rep protein, an AAV3 rep protein, an AAV4 rep protein, an AAV5 rep protein, an AAV6 rep protein, an AAV7 rep protein, an AAV8 rep protein; or a rep 78, rep 68, rep 52, rep 40, rep 68/78, and rep40/52; or a fragment thereof; or a protein from another source. Optionally, the rep and cap sequences are on the same genetic element in cell culture. There may be a spacer between the rep sequence and the cap gene. Optionally, the spacer is atgacttaaaccaggt, SEQ ID NO: 9. Any of these AAVhu68 or mutant AAV capsid sequences may be under the control of exogenous regulatory control sequences that direct their expression in the host cell.

In one embodiment of the invention, the cells are produced in a suitable cell culture (e.g., HEK 293) of cells. The methods for producing gene therapy vectors described herein include methods well known in the art, such as generating plasmid DNA used to produce gene therapy vectors, generating vectors, and purifying vectors. In some embodiments, the gene therapy vector is an AAV vector, and the plasmids generated are an AAV cis plasmid encoding an AAV genome and a gene of interest, an AAV trans plasmid containing the AAV rep and cap genes, and an adenoviral helper plasmid. The process of generating a vector may include method steps such as initiating cell culture, passage of cells, seeding cells, transfecting cells with plasmid DNA, post-transfection changing the medium to serum-free medium, and harvesting the cells containing the vector and the culture medium. The collected vector-containing cells and culture media are referred to herein as the crude cell harvest. In yet another system, gene therapy vectors are introduced into insect cells by infection with baculovirus vectors. For reviews of these production systems, see, for example, Zhang et al, 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the entire contents of which are incorporated herein by reference. Methods for making and using these and other AAV production systems are also described in the following U.S. patents, each of which is incorporated herein by reference in its entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 7094604; 7172893; 7201898; 7229823; and 7439065.

The crude cell harvest may then be subjected to method steps such as concentrating the vector harvest, diafiltering the vector harvest, microfluidizing the vector harvest, nuclease digesting the vector harvest, filtering the microfluidized intermediate, purifying the crude harvest by chromatography, purifying the crude harvest by ultracentrifugation, buffer exchange by tangential flow filtration, and/or formulating and filtering to yield bulk vector material.

A two-step high salt affinity chromatography purification followed by anion exchange resin chromatography is used to purify the vector drug and remove empty capsids. These methods are described in more detail in International Patent Application PCT/US2016/065970, filed December 9, 2016, and its priority documents, U.S. Patent Application Ser. Nos. 62/322,071, filed April 13, 2016, and 62/226,357, filed December 11, 2015, both entitled Scalable Method for Purifying AAV9, which are incorporated herein by reference. Methods for Purifying AAV8, International Patent Application No. PCT/US2016/065976, filed December 9, 2016, and its priority documents U.S. Patent Application Nos. 62/322,098, filed April 13, 2016, and 62/266,341, filed December 11, 2015, and rhlO, International Patent Application No. PCT/US16/66013, filed December 9, 2016, and its priority documents U.S. Patent Application Nos. 62/322,055, filed April 13, 2016, and 62/266,347, entitled Scalable Method for Purifying AAVrh10, also filed December 11, 2015, and AAV1, International Patent Application No. PCT/US2016/065974, filed December 9, 2016, and its priority documents, U.S. Patent Application Nos. 62/322,083, filed April 13, 2016, and 62/26,351, entitled Scalable Method for Purifying AAV1, filed December 11, 2015, incorporated herein by reference.

To calculate the empty and full particle content, the VP3 band volumes for a given sample (e.g., iodixanol gradient purified drug in the examples herein, where GC count = particle count) are plotted against GC loaded particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volumes of the drug being tested. The number of particles (pp) per 20 µl of loading is then multiplied by 50 to yield the particles (pp)/mL value. The pp/mL value divided by GC/mL yields the particle to genome copy ratio (pp/GC). Calculating pp/mL^C/mL yields the empty pp/mL value. The empty pp/mL value is divided by pp/mL and x100 to yield

- 32,048,431 percent empty particles.

In general, methods for analyzing empty capsids and AAV vector particles with packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6:13221330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsid, methods include subjecting the processed AAV stock to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% Tris-acetate in buffer, then running the gel electrophoresis until the sample material separates, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon. Then, antibodies against the AAV capsid are used as primary antibodies that bind to denatured capsid proteins, preferably a monoclonal antibody against the AAV capsid, most preferably a monoclonal antibody B1 against AAV-2 (Wobus et al., J. Virol. (2000) 74:9281-9293). Then, a secondary antibody is used, one that binds to the primary antibody and contains a means for detecting binding to the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently linked thereto, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. The binding detection method is used to semi-quantitatively detect binding between primary and secondary antibodies, preferably the detection method is capable of detecting radioactive isotope emissions, electromagnetic radiation or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g. DTT), and capsid proteins are separated on pre-prepared gradient polyacrylamide gels (e.g. Novex). Silver staining can be performed using SilverXpress (Invitrogen, California, USA) according to the manufacturer's instructions or another suitable staining method, i.e. SYPRO Ruby or Coomassie dyes. In one embodiment of the invention, the concentration of AAV vector genomes (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). Samples are diluted and digested with DNase I (or other suitable nuclease) to remove exogenous DNA. Following nuclease inactivation, samples are further diluted and amplified using primers and a TaqMan™ fluorogenic probe specific for the DNA sequence between the primers. The number of cycles required to achieve a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. Threshold cycle (Ct) values obtained for samples are used to determine the titer of vector genomes by normalizing it to the Ct value of the plasmid standard curve. Endpoint assays based on digital PCR can also be used.

In one aspect, an optimized q-PCR method is used that utilizes a broad-spectrum serine protease, such as proteinase K (e.g., commercially available from Qiagen). More specifically, the optimized qPCR genome titer assay is similar to the standard assay, except that after DNase I digestion, samples are diluted in proteinase K buffer and treated with proteinase K, followed by heat inactivation. Suitable samples are diluted in proteinase K buffer in an amount equal to the sample size. Proteinase K buffer can be concentrated up to 2-fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but can range from 0.1 mg/mL to about 1 mg/mL. The treatment step is typically performed at a temperature of about 55 °C for about 15 min, but it can be performed at a lower temperature (e.g., from about 37 °C to about 50 °C) for a longer period of time (e.g., from about 20 min to about 30 min), or a higher temperature (e.g., up to about 60 °C) for a shorter period of time (e.g., about 5 to 10 min). Similarly, heat inactivation is typically at a temperature of about 95 °C for about 15 min, but the temperature can be lowered (e.g., from about 70 to about 90 °C) and the time increased (e.g., from about 20 min to about 30 min). The samples are then diluted (e.g., 1000-fold) and subjected to the TaqMan assay as described in the standard assay.

Additionally, or as an alternative, droplet digital PCR (dcPCR) can be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers using dcPCR have been described. See, for example, M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub 2014 Feb 14.

Briefly, the method for separating rAAVhu68 particles having packaged genomic sequences from genome-deficient AAVhu68 intermediate particles comprises subjecting a suspension containing recombinant AAVhu68 viral particles and AAVhu689 intermediate capsid particles to rapid-release liquid chromatography, wherein the AAVhu68 viral particles and AAVhu68 intermediate particles are bound to a strong anion exchange resin equilibrated with

- 33 048431 suspended at pH 10.2, and subjected to a salt gradient by monitoring the eluate for ultraviolet absorbance at about 260 and about 280. Although less optimal for rAAV9hu68, the pH can range from about 10.0 to 10.4. In this method, complete AAVhu68 capsids are collected from the fraction that elutes when the A260/A280 ratio reaches the inflection point. In one example, for the affinity chromatography step, the diafiltered product can be applied to Capture Select™ Poros-AAV2/9 Affinity Resin (Life Technologies), which efficiently captures the AAV2/hu68 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while the AAV particles are efficiently captured.

III. Compositions and application.

The compositions provided herein comprise at least one parent rAAV (e.g., parent rAAVhu68 or a mutant parent rAAV) and an additional carrier, excipient, and/or preservative. Parent rAAV refers to a plurality of rAAV vectors that are the same, such as in the amounts described below in the discussion of concentrations and dosage units.

As used herein, the term carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such vehicles and agents for pharmaceutically active substances is well known in the art. Additional active ingredients can also be included in these compositions. The phrase pharmaceutically acceptable refers to molecular entities and compositions that, when administered to the host, do not cause an allergic or similar adverse reaction. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivering the vector genomes can be prepared for delivery either encapsulated in a lipid particle, liposome, vesicle, nanosphere, or nanoparticle, or the like.

In one embodiment, the composition is formulated for delivery to a subject, such as an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the composition. In another embodiment, the composition may be transported as a concentrate that is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

A suitable surfactant or combination of surfactants may be selected from nonionic surfactants that are non-toxic. In one embodiment of the invention, a surfactant with a bifunctional block copolymer terminated with primary hydroxyl groups is selected, such as, for example, Pluronic® F68 [BASF], also known as poloxamer 188, which has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e. nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic glyceride), polyoxy-10-oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment of the invention, the composition comprises a poloxamer. These copolymers are commonly referred to by the letter P (for poloxamer) followed by three digits: the first two digits x100 give the approximate molecular weight of the polyoxypropylene core, and the last digit x10 indicates the percentage of polyoxyethylene. In one embodiment of the invention, poloxamer 188 is selected. The surfactant may be present in the suspension in an amount of from about 0.0005% to about 0.001%.

Vectors are administered in amounts sufficient to transfect cells and provide sufficient levels of gene transfer and expression to provide a therapeutic effect without undesirable side effects, or medically acceptable physiological effects, as may be determined by those skilled in the art. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via the hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal and other parenteral routes of administration. Routes of administration may be combined if desired.

Dosages of the viral vector will depend primarily on factors such as the condition being treated, the age, weight, and health status of the patient, and thus may vary from patient to patient. For example, a therapeutically effective human dosage of a viral vector

- 34 048 431 is typically in the range of about 25 to about 1000 microliters, to about 100 ml of solution containing concentrations of about 1 x 10 ⁹ to 1 x 10 ¹⁶ viral vector genomes. The dosage will be adjusted to balance therapeutic benefit with any side effects, and such dosages may vary depending on the therapeutic application for which the recombinant vector is used. Expression levels of the transgene product can be monitored to determine the resulting dosing frequency of viral vectors, preferably AAV vectors containing the minigene. Optionally, dosing regimens similar to those described for therapeutic purposes can be used for immunization using the compositions of the invention.

The replication-defective virus compositions may be formulated as dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 10 ⁹ GC to about 1.0 x 10 ¹⁶ GC (for treating an average subject weighing 70 kg), including all integers or fractions within this range, and preferably from 1.0 x ^{10 12} GC to 1.0 x ^{10 14} GC for a human patient. In one embodiment of the invention, the compositions are formulated to contain at least 1 x 10 ⁹ , 2 x 10 ⁹ , 3 x 10 ⁹ , 4 x 10 ⁹ , 5 x 10 ⁹ , 6 x 10 ^{9 , 7 x 10 9 ,} 8 x 10 ⁹ or 9 x 10 ⁹ GC per dose, including all integers or fractions within this range. In another embodiment of the invention, the compositions are formulated to contain at least 1 x 10 ¹⁰ , 2 x 10 ¹⁰ , 3 x 10 ¹⁰ , 4 x 10 ¹⁰ , 5 x 10 ¹⁰ , 6 x 10 ¹⁰ , 7 x 10 ¹⁰ , 8 x 10 ¹⁰ or 9 x ^{10 10} GC per dose, including all integer or fractional values in this range. In another embodiment of the invention, the compositions are formulated to contain at least 1 x 10 ¹¹ , 2 x 10 11 , 3 x 10 ¹¹ , 4 x 10 ^{11 , 5 x 10 11} , 6 x 10 ¹¹ , 7 x 10 11 , 8 x 10 ¹¹ or 9 x 10 ¹¹ GC per dose, including all integer or fractional values in this range. In another embodiment of the invention, the compositions are formulated to contain at least 1 x 10 ¹² , 2 x 10 ¹² , 3 x 10 ¹² , 4 x 10 ¹² , 5 x 10 ¹² , 6 x 10 ¹² , 7 x 10 ¹² , 8 x 10 ¹² or 9 x 10 ¹² GC per dose, including all integer or fractional values in this range. In another embodiment of the invention, the compositions are formulated to contain at least 1 x 10 ¹³ , 2 x 10 13 , 3 ^x 10 ¹³ , 4 x 10 ¹³ , 5 x 10 ¹³ , 6 x 10 ¹³ , 7 x 10 ¹³ , 8 x 10 ¹³ or 9 x 10 ¹³ GC per dose, including all integer or fractional values in this range. In another embodiment of the invention, the compositions are formulated to contain at least 1 x 10 ¹⁴ , 2 x 10 ¹⁴ , 3 x 10 ¹⁴ , 4 x 10 ¹⁴ , 5 x 10 ¹⁴ , 6 x 10 ¹⁴ , 7 x 10 ¹⁴ , 8 x 10 ¹⁴ or 9 x 10 ¹⁴ GC per dose, including all integer or fractional values within that range. In another embodiment of the invention, the compositions are formulated to contain at least 1 x 10 ¹⁵ , 2 x 10 ¹⁵ , 3 x 10 ¹⁵ , 4 x 10 ¹⁵ , 5 x 10 ¹⁵ , 6 x 10 ¹⁵ , 7 x 10 ¹⁵ , 8 x 10 ¹⁵ or 9 x 10 ¹⁵ GC per dose, including all integer or fractional values in this range. In one embodiment of the invention, for use in humans, the dose may be in the range of 1 x 10 ¹⁰ to about 1 x 10 ¹² GC per dose, including all integer or fractional values in this range.

These above-mentioned doses can be administered in various volumes of carriers, excipients or buffer compositions in the range of about 25 to about 1000 microliters, including all numbers in this range, depending on the size of the area to be treated, the viral titer used, the route of administration and the desired effect of the method. In one embodiment, the volume of the carrier, excipient or buffer is at least about 25 μl. In one embodiment, the volume is about 50 μl. In another embodiment, the volume is about 75 μl. In another embodiment, the volume is about 100 μl. In another embodiment, the volume is about 125 μl. In another embodiment, the volume is about 150 μl. In another embodiment, the volume is about 175 μl. In yet another embodiment, the volume is about 200 μl. In another embodiment, the volume is about 225 μl. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In yet another embodiment, the volume is about 350 μL. In yet another embodiment, the volume is about 375 μL. In yet another embodiment, the volume is about 400 μL. In yet another embodiment, the volume is about 450 μL. In yet another embodiment, the volume is about 500 μL. In yet another embodiment, the volume is about 550 μL. In yet another embodiment, the volume is about 600 μL. In yet another embodiment, the volume is about 650 μL. In yet another embodiment, the volume is about 700 μL. In yet another embodiment, the volume is between about 700 and 1000 μL.

In certain embodiments, the dose may be in the range of about 1x10 ⁹ GC/g brain weight to about 1x10 ¹² GC/g brain weight. In certain embodiments, the dose may be in the range of about 3x10 10 GC/g brain weight to about 3x1011 GC/g brain weight. In certain embodiments, the dose may be in the range of about 1x10 9 GC/g brain weight to about 1x10 12 GC/g brain weight. In certain embodiments, the dose may be in the range of about 1x10 ¹⁰ GC/g brain weight to about 1x10 11 GC/g brain weight.

- 35 048431 of the invention, the dose may be in the range from about 5 x 10 ¹⁰ GC/g of brain mass to about 1.85 x 1011 GC/g of brain mass.

In one embodiment of the invention, the viral constructs can be delivered in doses of at least about at least 1 x 10 ⁹ GC to about 1 x 10 ¹⁵ , or about 1 x 1011 to 5 x 10 ¹³ GC. Suitable volumes for delivering these doses and concentrations can be determined by one of skill in the art. For example, volumes of 1 μL to 150 mL can be selected, with higher volumes being selected for adults. Typically, for newborn infants, a suitable volume is from about 0.5 mL to about 10 mL, for older infants, a volume of about 0.5 mL to about 15 mL can be selected. For pre-teens, a volume of about 0.5 mL to about 20 mL can be selected. For children, volumes of up to about 30 mL can be selected. For pre-pubertal adolescents and adolescents, volumes of up to about 50 mL can be selected. In still other embodiments of the invention, the patient may receive intrathecal administration in a selected volume of from about 5 ml to about 15 ml, or from about 7.5 ml to about 10 ml. Other suitable volumes and dosages may be determined. The dosage will be selected to balance the therapeutic benefit with any side effects, and such dosages may vary depending on the therapeutic application for which the recombinant vector is used.

The recombinant vectors described above can be delivered to host cells according to published methods. The rAAV, preferably suspended in a physiologically compatible carrier, can be administered to a human or non-human mammalian patient. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution comprising a saline solution, a surfactant, and a physiologically compatible salt or mixture of salts. Accordingly, the formulation is adjusted to a physiologically acceptable pH, such as in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of the cerebrospinal fluid is from about 7.28 to about 7.32, a pH in this range may be desirable for intrathecal delivery; whereas for intravenous delivery a pH of about 6.8 to about 7.2 may be desirable. However, other pH values within the broadest ranges and these subranges may be selected for other delivery routes.

In another embodiment of the invention, the composition includes a carrier, a diluent, an excipient and/or an adjuvant. Suitable carriers can be readily selected by one of skill in the art based on the indication for which the transfer virus is intended. For example, one suitable carrier includes saline, which can be prepared with various buffer solutions (e.g., sodium phosphate buffer). Other typical carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The buffer/carrier should include a component that prevents rAAV from adhering to the infusion tube but does not interfere with rAAV binding activity in vivo. A suitable surfactant or combination of surfactants can be selected from nonionic surfactants that are non-toxic. In one embodiment of the invention, a surfactant with a bifunctional block copolymer terminated with primary hydroxyl groups is selected, such as, for example, Pluronic® F68 [BASF], also known as poloxamer 188, which has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers, i.e. nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic glyceride), polyoxy-oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol, can be selected. In one embodiment of the invention, the composition comprises a poloxamer. These copolymers are commonly referred to by the letter P (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit x 10 indicates the percentage of polyoxyethylene. In one embodiment, poloxamer 188 is selected. The surfactant may be present in the suspension in an amount of from about 0.0005% to about 0.001%. In one example, the composition may comprise, for example, a buffered saline solution containing one or more of sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate 7H2O), potassium chloride, calcium chloride (e.g., calcium chloride 2H2O), dibasic sodium phosphate, and mixtures thereof in water. Accordingly, for intrathecal delivery, the osmolarity is in a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, e.g., emedicine.medscape.com/-article/2093316-overview. Optionally, for intrathecal delivery, a commercially available diluent can be used as a suspending agent or in combination with another suspending agent and other optional excipients. See, e.g., Elliott B® Solution [Lukare Medical]. In other embodiments, the composition

- 36 048431 may contain one or more permeation enhancers. Examples of suitable permeation enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9 lauryl ether or EDTA.

Optionally, the composition of the present invention may contain, in addition to rAAV and the carrier(s), other conventional pharmaceutical ingredients such as preservatives or chemical stabilizers. Suitable examples of preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The compositions of the present invention may comprise a pharmaceutically acceptable carrier, such as defined above. Accordingly, the compositions described herein comprise an effective amount of one or more AAVs suspended in a pharmaceutically acceptable carrier and/or mixed with suitable excipients, intended for delivery to a subject via injection, osmotic pump, intrathecal catheter, or other device or route of delivery. In one example, the composition is formulated for intrathecal delivery.

As used herein, the terms intrathecal delivery or intrathecal administration refer to the route of administration of drugs by injection into the spinal canal, more specifically into the subarachnoid space, so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital/intracisternal, and/or C1-2 puncture. For example, material may be administered for diffusion throughout the subarachnoid space via lumbar puncture. In another example, injection may be into the cerebellopontine cistern.

As used herein, the terms intracisternal delivery or intracisternal administration refer to the method of administering drugs directly into the cerebrospinal fluid of the cerebellomotor cistern magna, more specifically via suboccipital puncture or by direct injection into the pontocerebellar cistern or through an indwelling tube.

IV. Device and method for delivering a pharmaceutical composition into the cerebrospinal fluid.

In one aspect, the vectors provided in this document can be administered intrathecally using the method and/or device provided in this section and described further in Fig. 7. Alternatively, other devices and methods can be selected. The method includes the steps of inserting a needle into the patient's cerebellopontine cistern into the spine, connecting a section of flexible tubing to the proximal cannula of the spinal needle and the outlet of the valve to the proximal end of the flexible tubing, and after said insertion and connection steps and after the tubing can self-fill with the patient's cerebrospinal fluid, connecting a first vessel containing an amount of isotonic solution to the flushing inlet of the valve and then connecting a second vessel containing an amount of a pharmaceutical composition to the vector inlet of the valve. After connecting the first and second vessels to the valve, a path for fluid flow is opened between the vector inlet and the outlet of the valve, and the pharmaceutical composition is administered to the patient through the spinal needle, and after administration of the pharmaceutical composition, a path for fluid flow is opened through the flushing inlet and the outlet of the valve, and an isotonic solution is administered into the spinal needle to flush the pharmaceutical composition into the patient's body.

In another aspect, a device for intracisternal delivery of a pharmaceutical composition is proposed. The device includes a first vessel containing a certain amount of the pharmaceutical composition, a second vessel containing an isotonic solution, and a spinal needle through which the pharmaceutical composition can be injected from the device directly into the cerebrospinal fluid inside the patient's cerebellopontine cistern. The device further includes a valve having a first inlet port connected to the first vessel, a second inlet port connected to the second vessel, an outlet port connected to the spinal needle, and a Luer cone seal for controlling the flow of the pharmaceutical composition and the isotonic solution passing through the spinal needle.

As used in this document, the term computed tomography (CT) refers to an X-ray in which a three-dimensional image of the body's structure is created by a computer from a series of flat cross-sectional images taken along an axis.

The device or medical device 10, as shown in Fig. 7, includes one or more vessels 12 and 14, interconnected by a valve 16. The vessels 12 and 14 provide a fresh source of a pharmaceutical composition, drug, vector or similar substance and a fresh source of an isotonic solution, such as a saline solution, respectively. The vessels 12 and 14 can be a medical device of any shape that allows fluids to be administered to a patient.

- 37 048431

For example, each vessel 12 and 14 may be in the form of a syringe, cannula, or the like. For example, in the illustrated embodiment, vessel 12 is in the form of a separate syringe containing a quantity of pharmaceutical composition, and is referred to herein as a vector syringe. For example purposes only, vessel 12 may contain about 10 cc of pharmaceutical composition or the like.

Similarly, vessel 14 may be in the form of a separate syringe, cannula, or the like, which contains a certain amount of saline solution and may be referred to as a flush syringe. For example purposes only, vessel 14 may contain about 10 cc of saline solution.

Alternatively, the vessels 12 and 14 may be made in forms other than syringes and may be integrated into a single device, such as an integrated medical injection device, have a pair of separate chambers, one for the pharmaceutical composition and one for the saline solution. In addition, the size of the chambers or vessels may be provided as needed to contain the desired amount of liquid.

In the illustrated embodiment, the valve 16 is provided as a 4-way stopcock having a Luer cone pivot joint 18. The valve 16 connects the vessels 12 and 14 (i.e., the vector syringe and the flushing syringe in the illustrated embodiment), and the Luer cone pivot joint allows the path through the valve 16 to each of the vessels 12 and 14 to be closed or opened. Thus, the path through the valve 16 can be closed for both the vector syringe and the flushing syringe, or can be opened for a selected one of the vector syringe and the flushing syringe. As an alternative to a 4-way stopcock, the valve can be a 3-way stopcock or a fluid management device.

In the illustrated embodiment, the valve 16 is connected to one end of an extension tube 20 or similar fluid circuit. The tube 20 can be selected based on the desired length or internal volume. As an example only, the length of the tube can be from 6 to 7 inches.

In the illustrated embodiment, the opposite end 22 of the tube 12 is connected to a set 24 of T-shaped connector extensions, which in turn is connected to a spinal needle 26. As an example, the needle 26 may be a five-inch 22- or 25-gauge spinal needle. Additionally, as an option, the spinal needle 26 may be connected to a guide needle 28, such as a three-and-a-half-inch 18-gauge guide needle.

In use, the spinal needle 26 and/or the optional guide needle 28 can be advanced in the patient toward the pontocerebellar cistern. After the needle has been advanced, computed tomography (CT) images can be obtained that allow visualization of the needle 26 and/or 28 and the associated soft tissues (e.g., paraspinal muscles, bones, brainstem, and spinal cord). Correct placement of the needle is confirmed by observing the cerebrospinal fluid (CSF) in the needle cannula and visualizing the tip of the needle in the pontocerebellar cistern. Thereafter, a relatively short extension tube 20 can be attached to the inserted spinal needle 26, and then a 4-way stopcock 16 can be attached to the opposite end of the tube 20.

The above assembly can become a self-filling CSF of the patient. After this, the flushing syringe 14 pre-filled with a physiological solution is attached to the flushing inlet of the 4-way stopcock 16, and then the vector syringe 12 containing the pharmaceutical composition is attached to the vector inlet of the 4-way stopcock 16. After this, the outlet 16 of the stopcock is opened to the vector syringe 12, and the content of the vector syringe can be slowly introduced through the valve 16 and the assembled apparatus into the patient's body over a certain period of time. For example purposes only, this period of time can be approximately 1-2 minutes and / or any other time as desired.

After the contents of the vector syringe 12 have been injected, the rotary valves 18 on the stopcock 16 are rotated to a second position so that the stopcock 16 and the needle assembly can be flushed with a desired amount of saline solution using the attached pre-filled flushing syringe 14. As an example, 1 to 2 cc of saline solution can be used; although more or less can be used if necessary. The normal saline solution ensures that all or most of the pharmaceutical composition is forced through the assembled device to the patient and thus little or no pharmaceutical composition remains on the assembled device.

After the assembled device has been flushed with saline, the entire assembled device, including the needle(s), extension tubing, stopcock, and syringes, is slowly removed from the subject and placed on a surgical tray for transfer to a biohazard waste container or rigid container (for the needle(s)).

The principal investigator may conduct the screening process that ultimately leads to an intracisternal (IC) procedure. The principal investigator may describe the process, the procedure, the procedure itself, and the procedure.

- 38 048431 the insertion procedure and all potential safety risks so that the subject (or designated caregiver) is fully informed. Medical history, concomitant medications, physical examination, vital signs, electrocardiogram (ECG), and laboratory results are obtained or performed and provided to the neuroradiologist, neurosurgeon, and anesthesiologist for use in screening the subject's suitability for the IC procedure.

To allow sufficient time to verify eligibility, the following procedures may be performed at any time between the initial screening visit and up to one week prior to the study visit. For example, on day 0, magnetic resonance imaging (MRI) of the head/neck with and without gadolinium (ie, eGFR >30 mL/min/1.73 m2) may be obtained. In addition to the head/neck MRI, the investigator may determine the need for any further neck evaluation using flexion/extension studies. The MRI protocol may include T1, T2, DTI, FLAIR, and CINE protocol images.

Additionally, head/neck MRA/MRV may be obtained according to institutional protocol (i.e., subjects with a history of intra/transdural surgeries may be excluded or may require further testing (e.g., radionucleotide cisternography)), allowing adequate assessment of CSF flow and identification of possible blockage or absence of communication between CSF spaces.

The neuroradiologist, neurosurgeon and anesthesiologist ultimately discuss and determine the suitability of each subject for IC procedures based on all available information (scans, medical history, physical examination, labs, etc.). A preoperative anesthesia assessment may also be obtained from day -28 to day 1, which provides a detailed assessment of the airway, neck (shortened/thickened) and head range of motion (degree of neck flexion), keeping in mind the specific physiological needs of the MPS subject.

Prior to the IC procedure, the CT suite will confirm the presence of the following equipment and medications: Adult lumbar puncture (LP) kit (provided at each site); BD (Becton Dickinson) 22 or 25x3-7 gauge spinal needle (Quincke bevel); coaxial introducer needle used at the discretion of the interventionalist (for insertion of the spinal needle); small bore 4-way stopcock with Luer lock cone (Spin); set of T-connector extensions (tubing) with Luer lock cannula adapter, approximately 6.7 inches long; Omnipaque 180 (iohexol) for intrathecal administration; iodinated contrast for intravenous (IV) administration; 1% lidocaine for injection (if not provided in the adult LP kit); prefilled 10 cc saline (sterile) flush syringe; Radiopaque marker(s); surgical preparation equipment/razor; cushions/supports for proper positioning of the intubated subject; equipment for endotracheal intubation, general anesthesia machine, and mechanical ventilator; intraoperative neurophysiological monitoring (IONM) equipment (and necessary personnel); and a 10 cc syringe containing the vector; prepared and delivered to the CT/OR suite in accordance with the separate pharmacy formulary.

Informed consent for the procedure is confirmed and documented in the medical record and/or study file. Separate consent for the procedure is obtained from the radiologists and anesthesiologists in accordance with institutional requirements. The subject has intravenous access placed in the appropriate inpatient care unit in accordance with institutional guidelines (e.g., two IV access sites). Intravenous fluids are administered at the discretion of the anesthesiologist. At the discretion of the anesthesiologist and in accordance with institutional guidelines, the subject may be placed and endotracheally intubated under general anesthesia in the appropriate patient care unit, holding area, or surgical/CT suite.

A lumbar puncture is performed, first to remove 5 cc of cerebrospinal fluid (CSF) and then to inject intradermal contrast (Omnipaque 180) to aid in visualization of the pontine cistern. Appropriate maneuvers to position the subject may be performed to facilitate diffusion of contrast into the pontine cistern.

Intraoperative neurophysiological monitoring (IONM) equipment is attached to the subject. The subject is positioned on the CT scanner table in a prone or lateral decubitus position. Appropriate personnel should be present to ensure the safety of the subject during transport and positioning. If deemed appropriate, the subject may be positioned in a manner that allows neck flexion to the degree determined to be safe during the preoperative assessment and with normal neurophysiological monitor signals documented after positioning.

Relevant personnel can be confirmed as present and identified at the medical center: interventionalist/neurosurgeon performing the procedure; anesthesiologist and respiratory technologist(s); nurses and physician assistants; CT (or OR) technologists; specialist

- 39 048431 in neurophysiology; and the site coordinator. A time-out may be performed in accordance with Joint Commission/hospital protocol to verify the suitability of the subject, the procedure, the site, the location, and the presence of all necessary equipment in the room. The site lead investigator may then confirm to staff that he/she can proceed with subject preparation.

The subject's subcranial skin is shaved appropriately. Preoperative CT scans are obtained followed by a preprocedural planning CT scan with IV contrast if deemed necessary by the interventionalist to localize the target location and image the vasculature. Once the target site (pontocerebellar cistern) has been identified and the needle trajectory planned, the skin is prepared and draped using sterile technique according to established guidelines. A radiopaque marker is placed on the target skin area as directed by the interventionalist. The skin beneath the marker is anesthetized by infiltration with 1% lidocaine. A 22G or 25G spinal needle is then advanced to the pontocerebellar cistern with the option of using a coaxial introducer needle.

After needle advancement, CT images are acquired using the thinnest CT slice thickness possible with the equipment in place (ideally <2.5 mm). Serial CT images are acquired using the lowest possible radiation dose that provides adequate visualization of the needle and relevant soft tissues (eg, paraspinal muscles, bone, brainstem, and spinal cord). Correct needle placement is confirmed by observation of CSF in the needle cannula and visualization of the needle tip in the pontine cerebellar cistern.

The interventionist confirms that the vector syringe is located close to, but outside, the sterile field. Before handling or injecting the pharmaceutical composition into the vector syringe, personnel performing the procedure in the sterile field don gloves, a mask, and eye protection.

An extension tube is attached to the inserted spinal needle, which is then attached to the 4-way stopcock. After this apparatus is self-primed with the subject's CSF, a 10 cc pre-filled normal saline flush syringe is attached to the flush inlet of the 4-way stopcock. The vector syringe is then provided to the interventionalists and attached to the vector inlet on the 4-way stopcock.

After the outlet from the stopcock is opened to the vector syringe by placing the stopcock rotary knob in the first position, the contents of the vector syringe are injected slowly (over approximately 1-2 min), taking care not to apply excessive force to the syringe plunger during injection. After the contents of the vector syringe have been injected, the stopcock rotary knobs are rotated to the second position so that the stopcock and needle assembly can be flushed with 1-2 cc of saline solution using the attached pre-filled flush syringe.

When ready, the interventionist alerts the staff to remove the device from the subject. In one motion, the needle, extension tube, stopcock, and syringes are slowly removed from the subject and placed on a surgical tray for transfer to a biohazard waste container or rigid container (for the needle).

The needle insertion site is examined for signs of bleeding or CSF leakage and is prepared as directed by the investigator. The site is covered with gauze, surgical tape, and/or Tegaderm dressing as directed. The subject is then removed from the CT scanner and placed in a supine position on a stretcher. Appropriate personnel are present to ensure the safety of the subject during transport and positioning.

Anesthesia is discontinued and the patient is instructed to adhere to institutional guidelines for post-anesthesia care. Neurophysiological monitors are removed from the subject. The head of the stretcher on which the subject lies is to be elevated slightly (~30°) during recovery. The subject is transported to an appropriate post-anesthesia care unit in accordance with institutional guidelines. Once the subject has adequately regained consciousness and is stable, he/she will be admitted to the appropriate floor/unit for mandatory assessments as specified in the protocol. Neurological assessments will be performed as per protocol and the principal investigator will oversee the care of the subject in collaboration with hospital and study staff.

In one embodiment of the invention, the method for delivering a composition provided herein comprises the steps of: inserting a spinal needle into the pontocerebellar cistern of a patient; connecting a section of flexible tubing to the proximal cannula of the spinal needle and the outlet of the valve to the proximal end of the flexible tubing; after said insertion and connection steps and after the tubing is able to self-fill with the patient's cerebrospinal fluid, connecting a first vessel containing an amount of isotonic solution to the flushing inlet of the valve and then connecting a second vessel containing an amount of the pharmaceutical composition to the vector inlet of the valve; after connecting said

- 40 048431 of the first and second vessels with a valve open a path for fluid flow between the vector inlet and the outlet of the valve and administer a pharmaceutical composition to the patient through the spinal needle; and after injection of the pharmaceutical composition, open a path for fluid flow through the flushing inlet and the outlet of the valve and inject an isotonic solution into the spinal needle to flush the pharmaceutical composition from the patient. In a certain embodiment of the invention, the method further includes confirming the correct placement of the distal tip of the spinal needle within the pontocerebellar cistern before connecting the tube and the valve to the cannula of the spinal needle. In some embodiments of the invention, the step of confirming includes visualizing the distal tip of the spinal needle within the pontocerebellar cistern using computed tomography (CT) imaging. In some embodiments of the invention, the confirming step includes observing the presence of the patient's cerebrospinal fluid in the cannula of the spinal needle.

In the above-described method, the valve can be a shut-off valve with a Luer rotary joint, configured to rotate to a first position opening a flow from the vector inlet to the outlet, simultaneously blocking the flow through the flushing inlet, and to a second position opening a flow from the flushing inlet to the outlet, simultaneously blocking the flow through the vector inlet, and wherein the Luer rotary joint is located in said first position, when said pharmaceutical composition is injected into the patient, and is located in said second position, when said pharmaceutical composition is washed into said patient with an isotonic solution. In some embodiments of the invention, after introducing an isotonic solution into the spinal needle for washing the pharmaceutical composition into the patient, the spinal needle is removed from the patient with a tube, a valve and the first and second vessels connected to it in this assembly. In certain embodiments of the invention, the valve is a 4-way shut-off valve with a Luer rotary joint cone. In certain embodiments, the first and second vessels are separate syringes. In some embodiments, the T-shaped connector is located on the cannula of the spinal needle and connects the tube to the spinal needle. Optionally, the spinal needle includes a guide needle at the distal end of the spinal needle. The spinal needle can be a five-inch, 22- or 24-gauge spinal needle. In some embodiments, the guide needle is a 3.5-inch, 18-gauge guide needle.

In certain aspects, the method uses a device that consists of at least a first vessel for containing an amount of a pharmaceutical composition; a second vessel for containing an isotonic solution; a spinal needle through which the pharmaceutical composition can be withdrawn from the device directly into the cerebrospinal fluid inside the patient's cerebellopontine cistern; and a valve having a first inlet connected to the first vessel, a second inlet connected to the second vessel, an outlet connected to the spinal needle, and a Luer lock for controlling the flow of the pharmaceutical composition and the isotonic solution through the spinal needle. In some embodiments of the invention, the valve is a shut-off valve with a Luer joint configured to rotate to a first position opening the flow from the first inlet to the outlet while blocking the flow through the second inlet, and to a second position opening the flow from the second inlet to the outlet while blocking the flow through the first inlet. Optionally, the valve is a 4-way stopcock with a Luer lock cone. In certain embodiments, the first and second vessels are separate syringes. In some embodiments, the spinal needle is connected to the valve via a length of flexible tubing. A T-shaped connector may connect the tubing to the spinal needle. In some embodiments, the spinal needle is a 5-inch, 22- or 24-gauge spinal needle. In some embodiments, the device further comprises a guide needle connected to the distal end of the spinal needle. Optionally, the guide needle is a 3.5-inch, 18-gauge guide needle.

This method and this device may optionally be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices may be used for such intrathecal delivery.

In certain embodiments, the invention provides a composition that comprises the rAAVhu68.anti-HER2 antibody such that the AAV vectors carry nucleic acid expression cassettes encoding immunoglobulin constructs and regulatory sequences that direct the expression of such immunoglobulin in a selected cell. Once the vectors are introduced into the CNS, the vectors deliver the expression cassettes to the CNS and express the immunoglobulin protein constructs in vivo. The use of the compositions described herein as an antitumor method is described, as is the use of these compositions in antitumor regimens.

- 41 048431 max, which may optionally include delivery of one or more other antineoplastic or other active agents.

The composition may comprise one type of AAVhu68 vector, as described herein, that comprises an expression cassette for delivering an anti-tumor immunoglobulin construct in vivo. Alternatively, the composition may comprise two or more different AAV vectors, each having different expression cassettes packaged therein. For example, two or more different AAVs may have different expression cassettes that express immunoglobulin polypeptides that assemble in vivo to form a single functional immunoglobulin construct. In another example, two or more AAVs may have different expression cassettes that express immunoglobulin polypeptides for different targets, such as two cassettes provided for two functional immunoglobulin constructs (e.g., an anti-Her2 immunoglobulin construct and a second anti-tumor immunoglobulin construct). In another alternative embodiment, two or more different AAVs may express immunoglobulin constructs directed to the same target, wherein one of the immunoglobulin constructs has been modified to ablate FcRn binding and the second immunoglobulin construct retains its ability or has an increased ability to bind to FcRn. Such a composition may be suitable to simultaneously provide antibodies with increased retention in the brain region and antibodies for systemic delivery of the immunoglobulin construct.

Optionally, one or both of the immunoglobulin constructs described herein have enhanced ADCC activity. A regimen as described herein may comprise, in addition to one or more of the combinations described herein, an additional combination with one or more of an antitumor biological drug, an antitumor small molecule drug, a chemotherapeutic agent, immune enhancers, radiation, surgery, and the like. A biological drug as described herein is based on a peptide, a polypeptide, a protein, an enzyme, a nucleic acid molecule, a vector (including viral vectors), or the like.

Accordingly, the compositions described herein comprise an antitumor effective amount of one or more AAVhu68 suspended in a pharmaceutically acceptable carrier intended for delivery to a subject via injection, an osmotic pump, an intrathecal catheter, or other device or route of delivery. In one example, the composition is formulated for intrathecal delivery. As used herein, intrathecal delivery encompasses injection into the spinal canal, more particularly into the subarachnoid space. However, other delivery methods and pharmaceutically acceptable carriers for the AAV compositions may be selected including, for example, intracranial, intranasal, intracisternal, intracerebrospinal fluid delivery, including other suitable direct or systemic routes, i.e., the Ommaya reservoir.

The compositions can be formulated as dosage units to contain an amount of AAV that is in the range of about 1 x 10 ⁹ genome copies (GC) to about 5 x ^{10 13} GC (for treating an average subject weighing 70 kg). In one embodiment, a spinal puncture is performed in which about 15 ml (or less) to about 40 ml of CSF is removed, and in which the vector is mixed with the CSF and/or suspended in a compatible carrier and delivered to the subject. In one example, the vector concentration is about 3x10 ¹³ GC, but other amounts are possible, such as about 1x10 ⁹ GC, about 5x10 ⁹ GC, about 1x10 ¹⁰ GC, about 5x10 ¹⁰ GC, about 1x10 ¹¹ GC, about 5x10 11 ^GC , about 1x10 ¹² GC, about 5x10 ¹² GC, or about 1.0x10 ¹³ GC.

In one embodiment, the compositions described herein are used in a method for slowing tumor growth. In another embodiment, the compositions described herein are useful for reducing the size of a tumor in a subject. In another embodiment, the compositions described herein are useful for reducing the number of cancer cells in a non-solid malignancy. In another embodiment, a composition as provided herein is used in a method for increasing overall survival and/or progression-free survival in a patient. The anti-tumor immunoglobulin constructs are selected based on the neoplasm being treated. For example, to treat metastatic breast cancer to the brain, an expression cassette for an anti-HER antibody can be engineered into a recombinant AAV as described herein. Optionally, the AAV compositions as described herein are administered in the absence of an additional added pharmacological or chemical agent or other physical disruption of the blood-brain barrier. In combination therapy, the AAV-deliverable immunoglobulin construct described herein is administered before, during, or after the initiation of therapy with another agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during, and after the initiation of anticancer therapy. For example, the AAV can be administered between 1 to

- 42 048 431 days, preferably from 3 to 20 days, more preferably between 5 to 12 days before the start of radiation therapy. In another embodiment, the chemotherapy is administered simultaneously with or, more preferably, following AAV-mediated immunoglobulin (antibody) therapy. In other embodiments, the compositions of the invention can be combined with other biological agents, such as recombinant monoclonal antibody preparations, antibody-drug conjugates, or the like. In addition, combinations of different AAV-delivered immunoglobulin constructs, such as those described above, can be used in such regimens. Any suitable method or route can be used to administer the AAV1hu68.anti-HER2-containing composition as described herein, and optionally with co-administration of anti-tumor agents and/or other receptor antagonists. The regimens for the antitumor agent used in accordance with the present invention include any regimen that is believed to be optimally suitable for treating the neoplastic condition of the patient. Various malignant tumors may require the use of specific antitumor antibodies and specific anticancer agents, which will be determined for the patient based on his condition. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration. The dose of the antagonist administered depends on many factors, including, for example, the type of antagonists, the type of tumor and the severity of the tumor to be treated, and the route of administration of the antagonists.

It should be noted that singular terms include references to one or more. Therefore, singular terms, as well as the expressions one or more and at least one, are used interchangeably in this document.

The words comprise, contains, and comprising are to be interpreted inclusively and not exclusively. The words consist, consisting, and variations thereof are to be interpreted exclusively and not inclusively. Although various embodiments of the invention are described in the specification using the expressions comprising, in other circumstances a related embodiment of the invention is also intended to be interpreted and described using the expressions consisting of or consisting essentially of.

As used in this document, the term about means a variability of 10% (± 10%) from a given reference value, unless otherwise stated.

As used in this document, disease, disorder, and condition are used interchangeably to refer to an abnormal condition in a subject.

Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and are used with reference to published texts, which provide one skilled in the art with general guidance on many of the terms used in this invention.

The term expression is used herein in its broadest sense and includes the production of RNA or RNA and protein. With respect to RNA, the term expression or translation refers in particular to the production of peptides or proteins. Expression may be transient or may be stable.

As used herein, the term nAb titer is a measure of how much neutralizing antibody (e.g., anti-AAV nAb) is produced that neutralizes the physiological effect of its target epitope (e.g., AAV). Anti-AAV nAb titers can be measured as described, for example, by Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009. 199 (3): p. 381-390, which is incorporated herein by reference.

As used herein, an expression cassette refers to a nucleic acid molecule that contains a coding sequence, a promoter, and may include other expression control sequences. In certain embodiments, a vector genome may contain two or more expression cassettes. In other embodiments, the term transgene may be used interchangeably with the term expression cassette. Typically, such an expression cassette for creating a viral vector contains a coding sequence for a gene product described herein, flanked by viral genome packaging signals and other expression control sequences, such as those described herein.

The abbreviation sc in this context refers to self-complementary. The term self-complementary AAV refers to a construct in which the coding region carried by the nucleotide sequence of the recombinant AAV has been engineered to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated second-strand synthesis, the two complementary halves of the scAAV will associate to form a single unit of double-stranded DNA (dsDNA) that is ready for immediate replication and transcription. See, for example, D M McCarty et al, Self-complementary recombinant adeno

- 43 048431 associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis, Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patents 6,596,535; 7,125,717; and 7,456,683, the entire disclosures of each of which are incorporated herein by reference.

As used herein, the term operably linked refers to both expression regulatory sequences that are adjacent to a gene of interest and expression regulatory sequences that act in trans or remotely to control a gene of interest.

The term heterologous, when used in reference to a protein or nucleic acid, indicates that the protein or nucleic acid contains two or more sequences or subsequences that do not occur in the same relationship to each other in nature. For example, a nucleic acid typically produced recombinantly has two or more sequences from unrelated genes arranged to create a new functional nucleic acid. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from another gene. Thus, the promoter is heterologous with respect to the coding sequence.

A replication-defective virus or viral vector refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged within a viral capsid or envelope, wherein any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient, i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the viral vector genome does not contain genes encoding enzymes necessary for replication (the genome may be artificially created gutted - containing only the gene of interest flanked by signals necessary for amplification and packaging of the artificial genome), but these genes may be supplied during production. It is therefore considered safe for use in gene therapy because replication of progeny virions and infection by them cannot occur except in the presence of the viral enzyme required for replication.

In many cases, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endo- and exo-nucleases may also be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases may be selected to degrade single-stranded DNA and/or double-stranded DNA and RNA. Such steps may comprise a single nuclease or mixtures of nucleases directed to different targets and may be endonucleases or exonucleases.

The term nuclease resistant indicates that the AAV capsid is fully assembled around the expression cassette that is designed to deliver the gene into the host cell, and protects these packaged genomic sequences from degradation (cleavage) during nuclease incubation steps designed to remove contaminating nucleic acids that may be present during the manufacturing process.

As used herein, the term effective amount refers to an amount of the rAAV composition that delivers and expresses in target cells an amount of the gene product from the vector genome. An effective amount may be determined based on an animal model other than a human patient. Examples of suitable mouse models are described herein.

In certain embodiments, the rAAV or composition as provided herein excludes influenza antibodies or immunoglobulin constructs. In certain embodiments, the rAAV or composition as provided herein excludes the spinal muscular atrophy (SMA) gene or the SMN encoding sequence.

The term translation, as used herein, refers to the process in the ribosome in which a strand of mRNA controls the assembly of an amino acid sequence to form a protein or peptide.

As used in this specification and claims, the terms including, comprising, including and their variants include other components, elements, integers, steps and the like. On the other hand, the term consisting and its variants are exclusive with respect to other components, elements, integers, steps and the like.

It should be noted that singular terms include references to one or more, for example, an enhancer is understood to represent one or more enhancers. Therefore, singular terms, the expressions one or more and at least one are used interchangeably herein.

As described above, the term about when used to indicate a change in a numerical value means a change of ±10% unless otherwise stated.

The following examples are illustrative only and are not intended to limit the invention.

- 44 048431

Examples

In certain embodiments, the AAVhu68 capsid is noted to have a better yield than AAV9, which is also in clade F. One or both of the amino acid changes, glutamic acid (Glu) at position 67 and valine (Val) at position 157, can create this increased yield. In some embodiments, vectors having AAVhu68 capsids provide at least a 15% increase in packaged vector yield compared to AAV9-based vectors. In a comparison between AAVhu68 and AAVrh10, AAVhu68 was found to provide better transduction efficiency than AAVrh10 at a low dose (e.g., about 1 x 10 ⁹ ) following intracerebroventricular administration.

Example 1A. Definition of AAVHU68.

Tissue DNA was extracted from human tissue samples as a PCR template using QIAamp columns (Qiagen) according to the manufacturer's recommendations with the following modifications. Q5 DNA polymerase (Q5® Hot Start High-Fidelity 2X Master Mix, NEB) was chosen for its exceptionally high fidelity and reliable efficiency for the recovery of the full-length VP1 gene of potential AAVs in samples as described by Gao et al [Proc Natl Acad Sci USA, 2002 Sep 3, 99(18): 11854–11859 (Epub 21 Aug 2002)] with a primer set modified as follows: instead of AV1NS, the primer GCTGCGYCAACTGGACCAATGAGAAC, prm504 [SEQ ID NO: 7], and instead of the reverse primer AV2CAS, prm505:CGCAGAGACCAAGTTCAACTGAAACGA [SEQ ID NO: 8] were used. The PCR conditions were modified as follows:

PCR program:

Time (seconds) Cycle(s) 98 30 1 98 10 50 59 10 72 93 72 120 1

~3 kb PCR bands were excised from the gel; DNA was extracted using the QIAquick Gel Extraction Kit (Qiagen) and cloned into the Zero Blunt® TOPO® PCR Cloning Kit (Thermo Fisher Scientific). Plasmids were sequenced to obtain the full-length AAV VP1 gene. For most samples, at least three plasmids were fully sequenced and the consensus sequences were taken as the final AAV sequence for that sample.

The acquired nucleic acid sequence encoding the AAVhu68 vp1 capsid protein is shown in SEQ ID NO: 1. See also Figs. 2A-2C. The amino acid sequence of AAVhu68 vp1 is shown in Fig. 1 and SEQ ID NO: 2. Compared with AAV9, AAVhu31 and AAVhu32, two mutations (A67E and A157V) were identified as critical in AAVhu68 (circled in Fig. 1).

This amplification method also provides a spacer sequence between the vp1 coding sequence and the rep coding sequence. This coding sequence is: atgacttaaaccaggt, SEQ ID NO: 9. The coding sequence for rep52 from AAVhu68 is reproduced in SEQ ID NO: 3. The rep52 protein sequence is also reproduced in SEQ ID NO: 4.

The pAAV2/hu68 trans plasmid was then generated by inserting the hu68 VP1 gene into the pAAV2/9 backbone in place of the AAV9 VP1 gene to evaluate packaging efficiency, yield, and transduction properties. The pAAV2/9 plasmid contains the AAV2 5' and 3' ITRs flanking the capsid gene and is available from the Perm Vector Core [University of Pennsylvania, Phila, PA, USA, pennvectorcore.med.upenn.edu].

B. Characteristics of AAVHU68.

Although this phenomenon has not been previously observed or described in adeno-associated viral capsids, other proteins and peptides have been found to be susceptible, both in vivo and in vitro, to various chemical modifications. One of the most common modifications is asparagine deamidation, a spontaneous nonenzymatic reaction. In general, the half-lives of asparaginyl deamidation under physiological conditions (pH 7.4, 37°C) range between about 1 and 1000 days. A similar reaction sequence occurs in glutamine to glutamate residues, but these reactions are much

- 45 048431 slower than their analog asparagine.

In short peptides, the formation of cyclic intermediates is controlled by the primary sequence, while in proteins, secondary, tertiary, and quaternary structures have additional effects. Thus, the rate of deamidation of each protein amide is determined uniquely. Mass spectrometric identification of deamidated peptides is relatively straightforward because deamidation increases the mass of the intact molecule by +0.984 Da (the mass difference between the -OH and -NH2 groups). Since deamidation is a stable modification in the gas phase, MS/MS spectra can reveal the position of deamidation even in the presence of several potential deamidation sites.

Four AAVhu68 vectors were generated using one of four vector genomes that are not relevant to this study, each of which is produced using conventional triple-transfection methods in 293 cells. For a general description of these methods, see, e.g., Bell CL, et al., The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice., J Clin Invest. 2011;121:2427–2435. Briefly, a plasmid encoding the sequence to be packaged (the gene product expressed from the chicken β-actin promoter, intron, and growth hormone polyA) flanked by AAV2 inverted terminal repeats was packaged by triple transfection of HEK293 cells with plasmids encoding the AAV2 rep gene and the AAVhu68 cap gene and an adenoviral helper plasmid (pAdΔF6). The resulting AAV viral particles can be purified using CsCl gradient centrifugation, concentrated and frozen for later use.

Denaturation and alkylation: 2 μl of 1 M dithiothreitol (DTT) and 2 μl of 8 M guanidine hydrochloride (GndHCl) are added to 100 μg of thawed virus preparation (protein solution) and incubated at 90 °C for 10 min. Allow the solution to cool to room temperature, then add 5 μl of freshly prepared 1 M iodoacetamide (IAM) and incubate for 30 min at room temperature in the dark. After 30 min, quench the alkylation reaction by adding 1 μl of 1 M DTT.

Digestion: 20 mM ammonium bicarbonate, pH 7.5-8, is added to the denatured protein solution in a volume that dilutes the final concentration of GndHCl to 800 mM. Protease solution (trypsin or chymotrypsin) is added to a ratio of 1:20 protease to protein and incubated at 37°C overnight. After digestion, TFA is added to a final concentration of 0.5% to quench the digestion reaction.

Mass Spectrometry: Approximately 1 microgram of the combined digestion mixture was analyzed by UHPLC-MS/MS. LC was performed on an RSLCnano ULTIMATE 3000 system (Thermo Scientific). Mobile phase A was Milli-Q water with 0.1% formic acid. Mobile phase B was acetonitrile with 0.1% formic acid. The LC gradient was changed from 4% B to 6% B over 15 min, then to 10% B over 25 min (40 min total), and then to 30% B over 46 min (86 min total). Samples were loaded directly onto the column. The column size was 75 cm x 15 µm I.D. and it was packed with 2 µm C18 medium (Acclaim PepMap). LC is coupled to quadrupole-orbitrap mass spectrometry (Q-Exactive HF, Thermo Scientific) using a nanoflex electrospray ionization source. The column is heated to 35°C and electrospray is applied at 2.2 kV. The mass spectrometer is programmed to obtain tandem mass spectra from the main 20 ions. Full-scale MS resolution is set to 120,000 and MS/MS resolution to 30,000. Normalized impact energy is set to 30, automatic gain control to 1e5, maximum fill to 100 ms, and maximum MS/MS fill to 50 ms.

Data processing: Mass spectrometry raw data files were analyzed using BioPharma Finder 1.0 (Thermo Scientific). Briefly, all searches required 10 ppm precursor mass tolerance, 5 ppm fragment mass tolerance, tryptic digestion, up to 1 missed cleavage, fixed cysteine alkylation modification, variable methionine/tryptophan oxidation modification, asparagine/glutamine deamidation, phosphorylation, methylation, and amidation.

In the following table, T refers to trypsin and C refers to chymotrypsin.

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Modification enzyme T T T T WITH WITH WITH WITH T T T % Coverage 93.6 92 93, 1 92.5 90, 2 89, 7 91, 1 88, 9 98.9 97 94, 6 92, 4 + Deamidation (Desamid) -N35 N57+ Des amide 87.6 95, 5 89, 3 88.2 90, 5 96, 3 86, 4 84, 8 100, 0 100, 0 99, 0 92, 7 N66+ Des amide 4.7 N94+ Des amide 11.3 u, 9 and, 0 5.3 and, 6 y, 4 u, 8 5,6 5.0 ID 5.4 16, 0 N113+ Des amide 1,8 -N253+ Des amide 17.7 22, 0 21, 1 15,0 17, 0 22, 6 20, 5 15, 6 4.2 5.5 Q259+ Des amide 35.2 25, 6 21, 0 35, 4 26, 3 20, 9 9.2 -N270+ Des amide 16.4 25, 1 23, 2 16.6 15, 9 24, 9 23, 5 16, 1 0,2 -N304+ Des amide 2.6 2.9 2.8 1,3 2,5 2.8 2.9 1,3 16.6 10.3 -N314+Desamid 6.5 N319+ Des amide 0,3 2.8 2.8 0,2 2.9 2.8 0,2 N329+ Des amide 72.7 85, 6 89, 1 86.8 71, 0 87, 2 88, 7 84, 7 85.5 79.4 78, 9 91, 8 N336+ Des amide 30, 8 9.3 100, 0 31, 0 9.2 95, 7 -N409+ Des amide 21.3 22, 9 23, 9 24,0 22, 0 23, 4 24, 7 24, 2 N452+ 98.8 99, 99, 100, 98, 97, 98, 95, 98.2 68.7 67, 49,

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Desamid 7 2 0 9 3 1 2 4 4 N477+ Desamid 4,4 4.3 4.3 2.6 4,5 4,4 4.3 2.6 0.8 N512+ Desamid 97.5 97, 9 95, 3 95.7 92, 2 91, 8 99, 2 96, 1 99.7 98.2 87, 9 75, 7 -N515+ Desamid 8.2 21, 0 16, 0 8.3 21, 0 16, 5 0,0 2,5 3.0 15, 1 -Q599+ Desamid 4.0 15, 4 y, 1 13.6 4.0 15, 5 y, 0 13, 8 15.8 N628+ Desamid 5.3 5,6 5.4 0,0 5.4 0,0 N651+ Desamid 0.9 1,6 1,6 0,5 N663+ Desamid 3,4 3.5 3.7 3,4 0,0 3,4 3.6 N709+ Desamid 0,6 0.8 20, 2 0,6 0,6 0,8 19, 8 0,6 0,3 1,3 0,1 0,2 N735 25,0 42.7 21, 7 + Acetylation (Ac): K332+Ac 100, 0 ~K693+Ac 13,0 13, 5 -Kbbb+Ace 93.8 ~K68+Ac 59, 2 + Isomerization (Iso): I97+Iso 0,5 0,4 0,4 0,2 0,5 0,4 0,2 0107+Iso 0,3 0,3 0,3 I384+Iso 0.8 0.9 + Phosphorylation

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(Phos) 8149+Phos 5.8 5.7 5.2 9.8 5.7 5.9 5.2 9.9 -S499+ Phos 30.6 -T569+ Phos 0.9 -S586+ Phos 3.6 + Oxidation ~W23+Okhc 4.7 5.5 4.8 5.5 W247+Okhc 1,5 0,4 0,7 1,4 W247+Okhc to kynurenine 0,1 0,1 W306+Okhc 0,7 0.9 1,6 1,8 0,7 1,0 1,6 1,8 W 3 O6+Oxidation to kynurenine 0,3 0,3 M404+Oxide 0,1 0,2 0,1 0,2 M436+Oxide 4.9 y, 2 23,0 4.8 y, 2 22, 6 -M518+ Oxide 29.9 1,5 10.6 29, 9 1,5 y, 5 -M524+ Oxide 18.8 31, 6 52, 7 18, 4 31, 1 52, 5 14, 2 M559+Oxide 19,0 21, 6 19, 6 20.9 19, 6 21, 3 20, 1 20, 9 -M605+ Oxide 12.2 15, 2 12, 8 14, 8 W619+Okhc 1,0 0,6 1,5 1,0 0,6 1,5 W619+Oxidation 20, 3 -M640+ Oxide 23.5 64, 2 24, 6 22, 4 21, 1 25, 6 W695+Okhc 0,3 0,4 0,4 0,3 0,4 0,4

The major asparagine residues (N94, N253, N270, N304, N409, N477, and Q599) also show deamidation levels of up to ~20% across different lots. Deamidation levels were initially identified using trypsin digestion and verified using chymotrypsin digestion.

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Example 2. AAVHU68 vector output.

AAVhu68 and AAV9 vectors carrying various tags such as GFP and LacZ were generated and evaluated. Each vector was generated using the triple transfection method in 293 cells as described by Gao et al. [Gao, Guang-Ping et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proceedings of the National Academy of Sciences 99.18 (2002): 11854–11859.]

A. Preparation of PAAVHU68 trans-plasmid.

The nucleic acid sequence encoding the vp1 capsid protein is presented in SEQ ID NO: 1.

The pAAV2/hu68 trans plasmid was generated by inserting the hu68 VP1 gene into the pAAV2/9 backbone in place of the AAV9 VP1 gene to evaluate packaging efficiency, yield, and transduction properties. The pAAV2/9 plasmid contains the AAV2 5' and 3' ITRs flanking the capsid gene and is available from the Penn Vector Core [University of Pennsylvania, Phila, PA, USA, pennvectorcore.med.upenn.edu].

B. AAVHU68 vector output.

293 cells were cultured and maintained in DMEM, 1X (Dulbecco's modification of Eagle's minimum essential medium) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate supplemented with 10% fetal bovine serum in an atmosphere of 5% _CO2 at 37°C. Transfections were performed as described by Gao et al. [Gao, Guang-Ping et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proceedings of the National Academy of Sciences 99.18 (2002): 11854–11859.] with the vector plasmid replaced with pAAV2/hu68 or pAAV2/9. CB7.CI.ffLuciferase.RBG was used as the transgene (expression cassette). Transfected cells were then cultured in 6-well plates. Total cell lysate as well as supernatant were collected for virus quantification by TaqMan assay (Applied Biosystems) using probes and primers targeting the polyA region of the rabbit beta-globin transgene (expression cassette) as described by Gao et al. [Gao, Guangping, et al. Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo. Human gene therapy 11.15 (2000): 2079–2091]. The yields of six pAAV2/9 and six pAAV2/hu.68 plasmids were compared in a 6-well plate against each other in terms of both supernatant and total lysate titer. Each plasmid was obtained from a single bacterial colony.

The yield of AAVhu68 was similar to that of AAV9 in terms of total lysate (Figure 3A, n=6, p=0.42). However, in the supernatant, the yield of AAVhu68 was significantly higher than that of AAV9 (Figure 3B, n=6, p=0.0003). Thus, AAVhu68 was demonstrated to be a superior vector compared to AAV9 in terms of production, since the supernatant was harvested for cell-stack flask scale culture and virus production.

Example 3. In vivo transduction of AAVHU68.LACZ.

AAVhu68.CB7.nLacZ (also referred to as AAVhu68.LacZ) was constructed by inserting the coding sequence for nuclear-localized bacterial β-galactosidase (nLacZ) and then produced as described in Example 2. To evaluate the packaging efficiency, yield, transduction properties, transduction efficiency, and tropism of AAVhu68 in vivo, mice were injected with ^5x1ο11 copies of the AAVhu68.LacZ vector genome via different routes of administration, such as intravenous, intramuscular, and intranasal administration. Muscle, lung, liver, and heart were collected after mice were sacrificed two weeks after vector injection. Frozen sections of each organ were prepared, processed, and analyzed according to a conventional protocol detecting LacZ gene expression [Bell, Peter, et al. An optimized protocol for detection of E. coli β-galactosidase in lung tissue following gene transfer. Histochemistry and cell biology 124.1 (2005): 77–85]. Positive staining for LacZ, shown in blue (Fig. 4A–4C), indicates successful transduction of AAVhu68.

As shown in Fig. 4A, after the vectors were administered to mice by intravenous injection (i.v.), all organs tested (heart, liver, lung, and muscle) showed AAVhu68 transduction, while a tropism favoring the heart and liver over the lung and muscle was observed. After the vectors were administered to mice by intramuscular injection (i.m.), the heart, liver, and muscle showed a high rate of AAVhu68 transduction, while no detectable transduction was observed in the lung. When intranasal administration was performed, diffuse transduction was observed in the heart, liver, muscle, and lung.

These results showed that AAVhu68 exhibited high transduction efficiency and broad tissue/organ tropism.

Example 4. In vivo transduction of AAVHU68.GFP versus AAV9.GFP.

AAVhu68.GFP and AAV9.GFP were generated by inserting the gene encoding green fluorescent protein (GFP) as genes, which were then prepared as described in Example 2. To evaluate the packaging efficiency, yield, transduction properties, transduction efficiency, and tropism of AAVhu68 and AAV9 in vivo, mice were injected with AAVhu68.GFP or AAV9.GFP at doses of 1x 10 ¹⁰ GC or 1x1011 GC. Brain, muscle, lung, liver, and heart were collected after mice were sacrificed two weeks after vector administration. Frozen sections of each organ were prepared and processed for visualization

- 50 048431 GFP expression was detected as described by Wang et al [Wang L, et al., Hum Gene Ther. 2011 Nov; 22(11):1389-401; Wang L, et al., Mol Ther. 2010 Jan; 18(l):126-34]. Positive staining for GFP, shown in green (Fig. 5A-5C and Fig. 6A-6D), indicates successful transduction of the tested vectors.

Brain slices from different regions (hippocampus, motor cortex and cerebellum) of mice injected with vectors intracerebroventricularly were examined. Transduction of AAV vectors was observed in all hippocampal samples tested except one of the mice injected with 1 x 10 ¹⁰ GC AAV9.GFP. Better transduction of AAVhu68.GFP compared to AAV9 was observed in the motor cortex. Moreover, transduction of AAVhu68.GFP was observed in the cerebellum when mice were injected with only 1 x 10 ¹¹ GC vector. Thus, AAVhu68 demonstrated higher transduction efficiency as well as broader tropism in the brain compared to AAV9.

In the next experiment, various organs such as liver, kidney, heart and pancreas were harvested from mice injected intravenously with AAVhu68.GFP and the organs were prepared and processed as described by Wang et al. [Wang L, Calcedo R, Bell P, Lin J, Grant RL, Siegel DL, Wilson JM, Hum Gene Ther. 2011 Nov; 22(11):1389-401; Wang L, Calcedo R, Wang H, Bell P, Grant R, Vandenberghe LH, Sanmiguel J, Morizono H, Batshaw ML, Wilson JM, Mol Ther. 2010 Jan; 18(1):126-34]. A positive GFP signal, shown in green, indicates successful transduction of the indicated AAV vectors. Bright field images shown in black and white were taken to determine organ morphology, while the corresponding red fluorescent channel was performed as a negative control.

A strong positive signal, shown in green, was observed in the liver, with kidney, heart and pancreas also showing transduction of the vector, indicating broad tissue/organ tropism of the AAVhu68 vector.

Example 5. Yield and in vivo transduction of AAV vectors with A67E and A157V mutations.

To improve the yield and/or packaging efficiency of a recombinant adeno-associated virus (rAAV) vector, an AAV capsid gene was engineered to express the vp1 protein with Glu at amino acid position 67 and/or Val at amino acid position 157 of AAV vectors such as AAV9, AAVhu31, and AAVhu32, with the amino acid residue numbering based on AAVhu68 [SEQ ID NO: 5].

The indicated AAV vectors were prepared and the yield of each vector was assessed according to Example 2. In vivo transduction efficiency and tissue/organ/site tropism were further assessed using conventional methods such as those illustrated in Example 3.

Example 6. Intrathecal AAVHU68.CMV.PI.HTRASTUZUMAB.SV40 for the prevention of HER2+ human breast cancer brain metastases.

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AAV9 Adeno-associated virus 9 AAV9. trastuzumab AAV9.CMV.PI.htrastuzumab.SV40 (AAV9 carrying the trastuzumab expression cassette) VSA Bicinchoninic acid assay VSVM Breast cancer metastases to the brain CI Chimeric intron CMV (promoter) Cytomegalovirus immediate early enhancer/chicken beta-actin promoter CMS Cerebrospinal fluid ccPCR Droplet digital polymerase chain reaction DNA Deoxyribonucleic acid GC Genome Copies GLP (Good Laboratory Practices) Good Laboratory Practice GTP (Gene Therapy Program) Gene therapy program HER2 Human epidermal growth factor receptor 2 AAVhu68 Adeno-associated virus serotype hu68 AAVhu68.trastuzumab hu68.CMV.PI.htrastuzumab.SV40 (AAVhu68 carrying the trastuzumab expression cassette) ICV Intracerebroventricular ID Identification number IT Intrathecal mAt Monoclonal antibody MED Minimum required dose n Number of animals PBS Phosphate buffered saline qPCR Quantitative polymerase chain reaction RAGl ^-/ “ Knockout of recombination activating gene 1 RAG1 Recombination activating gene 1 rBG Rabbit β-globin poly-A sequence RPM Revolutions per minute SD Standard deviation SOP Standard Operating Procedure SV40 (poly-A signal) Simian virus 40 polyadenylation signal

A. Summary.

The aim of this study was to test the therapeutic efficacy of AAVhu68.CMV.PI.htrastuzumab.SV40 (AAVhu68.trastuzumab), a recombinant adeno-associated virus serotype AAVhu68 containing the trastuzumab expression cassette, for the prevention of HER2+ human breast cancer brain metastasis in a mouse xenograft model. Trastuzumab (Herceptin®, Roche) is a humanized monoclonal antibody (mAb) directed against HER2 that prolongs patient survival when administered intravenously with chemotherapy for the treatment of systemic HER2+ disease. However, the blood-brain barrier prevents intravenous Herceptin® from entering the central nervous system, rendering it ineffective in treating HER2+ breast cancer brain metastasis. Several clinical cases show that intrathecally administered Her

- 52 048431 ceptin® may prolong survival in patients with HER2+ leptomeningeal disease or halt progression of HER2+ focal metastases [J. C. Bendell, et al, Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma. Cancer. 97, 2972-2977 (2003); D. J. Slamon, et al., Use of Chemotherapy plus a Monoclonal Antibody against HER2 for Metastatic Breast Cancer That Overexpresses HER2. N. Engl. J. Med. 344, 783-792 (2001), M. A. Cobleigh, et al, Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J. Clin. Oncol. 17, 2639–2648 (1999), Zagouri F, et al, (2013). Intrathecal administration of trastuzumab for the treatment of meningeal carcinomatosis in HER2-positive metastatic breast cancer: a systematic review and pooled analysis. Breast Cancer Res Treat, 139(1): 13–22., Bousquet G, et al. (2016). Intrathecal Trastuzumab Halts Progression of CNS Metastases in Breast Cancer. J Clin Oncol. 34(16 ):e151–155]. However, CSF is rapidly renewed, likely compromising the therapeutic effect of IT Herceptin® due to the widely variable pharmacokinetic profile of CSF. The goal of AAVhu68.trastuzumab treatment is to prevent the onset, slow the growth, improve survival, or increase the clinical quality of life associated with HER2+ BCM (breast cancer brain metastases) by providing localized, long-term expression of AAVhu68.trastuzumab within the brain parenchyma itself.

AAVhu68.trastuzumab was administered at four different doses (1.00x101°, ^3.00x1010 , 1.00x10, and 3.00x10 GC/animal) by intracranial ventricular injection (ICV) into 6- to 9-week-old RAG1 ^-/- mice. BT474.M1.ffluc cells, a HER2+ human ductal carcinoma cell line, were implanted at least 21 days later. Mice were observed daily and sacrificed at the endpoint of the study. Brain tissue was collected at necropsy for tumor volume measurement. It was concluded that prophylactic ICV administration of AAVhu68.CMV.PI.htrastuzumab.SV40 in the RAG1 ^-/- xenograft model of HER2+ BC brain metastases resulted in a significant reduction in tumor volume at all doses tested in this experiment. Overall, these results demonstrated the potential therapeutic efficacy of AAVhu68.trastuzumab to improve survival in patients with HER2+ BCBM.

B. The aim of this study was to investigate the minimum required dose (MED) of AAVhu68.trastuzumab for tumor prevention in a RAG1-/- xenograft model with HER2+ HCMV by examining tumor volume. The vector is AAVhu68.CMV.PI.htrastuzumab.SV40 or AAVhu68.trastuzumab.

qPCR titer: 7.38x1013 GC/ml.

Endotoxin: <2.0 EU/ml.

Purity: 100%.

Phosphate buffered saline (PBS) (no treatment control).

The tumor preventive potential of AAVhu68.trastuzumab was assessed using a RAG1-/- HER2+ HCVD xenograft mouse model. The immunodeficient mouse model allows the growth of human-derived orthotopic tumors in mice without rejection by the mouse immune system. In addition, the RAG1-/- mouse lacks its own IgG, allowing quantification of trastuzumab using protein A ELISA.

Study design

No. groups Treatment Dose (GC/mice) Genotype (η) Dose volume (µl) RO A Tumor cells Implantation 1 AAVhu68.trastuzu mab ¹⁰ RAGl ^-/ “ (10) 5 ICV 21 days after treatment 2 AAVhu68.trastuzu mab 3,OX1O ¹⁰ RAGl“ ^/_ (10) 5 ICV 3 AAVhu68.trastuzu mab ¹¹ RAGl ^-/ “ (10) 5 ICV 4 AAVhu68.trastuzu mab 3.0Χ10 ^π RAGl ^-/ “ (10) 5 ICV

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5 PBS Without treatment RAGl ^-/ “ (U) 5 ICV

The test drug and negative control were diluted with sterile phosphate-buffered saline (PBS) to the appropriate concentration. The vector was injected ICV into the left lateral ventricle.

Intrathecal delivery of AAV can be accomplished using a variety of routes to access the CSF. The ICV route was chosen because it is minimally invasive and does not require a surgical procedure in mice (compared to the pontine cistern route, which requires an incision in the skin and neck muscles). Our laboratory and others have previously shown that a single injection of AAV9 vector into the cerebrospinal fluid (ICV or pontine cistern) in both mice and large animals affects neurons throughout the brain [Dirren at al. (2014). Intracerebroventricular Injection Of Adeno-Associated Virus 6 And 9 Vectors For Cell Type-Specific Transgene Expression In The Spinal Cord. Hum. Gene. Therapy 25, 109-120, Snyder et al. (2011). Comparison Of Adeno-Associated Viral Vector Serotypes For Spinal Cord And Motor Neuron Gene Delivery. Hum. Gene Ther 22, 1129-1135, Bucher et al. (2014). Intracisternal Delivery Of AAV9 Results In Oligodendrocyte And Motor Neuron Transduction In The Whole Central Nervous System Of Cats. Gene Therapy 21, 522-528, Hinderer et al. (2014). Intrathecal Gene Therapy Corrects CNS Pathology In A Feline Model Of Mucopolysaccharidosis I. Mol Ther: 22, 2018-2027].

C. Implantation of RAG1-/- tumor cells into mice.

To generate a xenograft mouse model for HER2+ BCVM, a firefly luciferase-transduced HER2+ human ductal carcinoma cell line, BT474-M1.ffluc, was used. Mice were anesthetized with ketamine/xylazine for the injection procedure. The scalp and neck hair was clipped. Sustained-release IV-β estradiol pellets (1.7 mg, 90-day release, Innovative Research of America) were implanted subcutaneously in the dorsum of the neck and re-administered every 90 days during the study. Mice were mounted in a stereotaxic apparatus. Exposed skin was cleaned with povidone-iodine and 70% ethanol. A 1-cm anteroposterior incision was made over the top of the skull. Bregma was identified. The pneumatic drill, positioned at the vertex, was then moved 0.8 mm posteriorly and 2.2 mm to the left of bregma, where a burr hole had been drilled into the skull. A 25 μl Hamilton syringe was filled with 5 μl of tumor cell suspension (100,000 cells in a 50:50 ratio of MatriGel®:PBS). The needle was advanced to bregma and moved to the coordinates indicated above before penetrating 4.0 mm into the brain parenchyma. The needle was then lifted back up 1.0 mm along the needle trajectory to create a pocket into which tumor cells were injected. The needle was left in place for 5 min. Then, 5 μl of cell suspension was injected over 10 min using a motorized injection device. The needle was left in place for 5 min after the end of the injection and then slowly removed. The scalp incision was sutured with Vicryl 4.0 and mice received 15 mg/kg enrofloxacin (Bayer) subcutaneously in sterile PBS along with 0.3 mg/kg buprenorphine in sterile PBS.

Mice were monitored daily. When moribund, mice were sacrificed by _CO2 overdose followed by cervical dislocation. At necropsy, the brain was isolated and sectioned coronally through the injection needle track in the tumor.

Tumor volume: Tumor diameter measurements at day 35 were performed with Vernier digital calipers (Thermo-Fisher). Brains were harvested at autopsy. Peeling along the injection needle track in the tumor was used to separate the tumor from the surrounding brain tissue. Tumor diameter was then measured in 3 dimensions (x, y, and z), and tumor volume was calculated as the volume of an ellipsoid, 4/3 * π * x/2 * y/2 * z/2. The right cerebral hemisphere, the hemisphere contralateral to the site of vector injection and tumor implantation, was preserved in formalin. Dissected tumors were pooled by dose cohort and preserved in formalin. Tumor volume comparisons were performed using the Mann-Whitney test in GraphPad Prism 7.

D. Results.

Tumor Volume: To determine whether IT tumor prophylaxis with AAVhu68.trastuzumab slows tumor growth, we measured tumor diameter 35 days after implantation. The median tumor volume from the group that received the highest dose of AAVhu68.trastuzumab tumor prophylaxis (0.4 ^mm3 , n=10) was significantly smaller than that of mice that did not receive any treatment (26.1 ^mm3 , n=9). All mice that received lower doses of AAVhu68.trastuzumab had significantly smaller tumors compared to no treatment. The median tumor volume in mice receiving ^1.00x1010 GC/mouse was calculated to be statistically the same as the median tumor volume in mice receiving ^3.00x1010 GC/mouse (p=0.6029). It should be noted that two mice in Group 1, one mouse in Group 2, three mice in Group 3, and three mice in Group 4 had no macroscopically visible tumor at necropsy.

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Group Dose (GC/mouse) Number of mice Median tumor volume (mm ³ ) p-value compared to no treatment 1 ¹⁰ 9* 6.4 0,0375 2 3.00X10 ^w 10 8.1 0,0053 3 ¹¹ 9* 1,3 0,0026 4 3.00Χ10 ^η 10 0,4 < 0.0001

*One animal in each of these groups was sacrificed prior to the scheduled necropsy date and was therefore not included in the analysis.

At all doses, IT administration of AAVhu68.trastuzumab resulted in a significantly lower median tumor volume at D35 post-implantation when administered prophylactically in the RAG1 mouse xenograft model of HER2+ BCBM, which utilizes the HER2+ BT474.M1 human ductal carcinoma cell line. The MED of AAVhu68.trastuzumab measured in this study was 1.00 x 10 ¹⁰ GC/mouse.

Example 7. Yield and purity of AAVHU68 vector products.

To compare the yield and/or purity of recombinant adeno-associated virus (rAAV) vector production with different capsids, two different sets of vectors with different capsids were generated and prepared, including AAVhu68, AAV8triple, AAV8, and AAV9.

Briefly, one set of vectors having the indicated capsid and vector genome containing cytomegalovirus (CMV) promoter, firefly luciferase coding sequence and SV40 poly-A (CMV.ffLuciferase.SV40) were generated and the yield of each vector was assessed at small scale. The results showed that AAV9 vectors presented the highest yield, while AAVhu68 vector was in second place (Fig. 8A). AAV8 and AAV8 triple vectors also provided yields above ^4x1013 GC (Fig. 8A).

Another set of vectors having the indicated capsid and a vector genome containing the CMV promoter, intron, coding sequence of immunoadhesin (201Ig IA) and SV40 poly-A (CMV.PI.201Ig IA.SV40) were obtained and assessed for yield and purity of each vector at mega-scale according to routine methods. The results are shown in Fig. 8B and 9.

Similar to the small scale yields, AAV9 vectors presented the highest yield of about ^5.7x1014 GC, while AAVhu68 vector was second with about ^3.8x1014 GC (Fig. 8B). AAV8 vectors provided a yield of about ^3.6x1014 GC and AAV8tirple about ^1.8x1014 GC (Fig. 8B). The purity of the tested preparations was comparable, ranging from about 97.4% to about 98.6%.

Example 8. RAAV vectors in male RAG KO mice.

Gene expression was tested in vivo using rAAV vectors containing different capsids, including AAVhu68, AAV8triple, AAV8, and AAV9, and expressing a secreted transgene product, 201Ig IA.

Male 6-8 week-old RAG KO mice (n=5/group) were injected intramuscularly into the gastrocnemius muscle with either 3x ¹⁰¹¹ GC/mouse or 3x ¹⁰¹⁰ GC/mouse of the test vector using a Hamilton syringe. Serum was collected weekly from mice injected with vectors expressing secreted proteins by submandibular blood collection tubes. Transgene expression levels were measured in serum by ELISA as described in Greig et al., Intramuscular Injection of AAV8 in Mice and Macaques Is Associated with Substantial Hepatic Targeting and Transgene Expression, PLoS One. 2014 Nov 13;9(11):e112268. doi: 10.1371/journal.pone.0112268. eCollection 2014.

As shown in Fig. 10A and 10B, AAVhu68, AAV8, and AAV9 vectors expressed the transgene at similar levels, and AAV8triple vector expressed it better after i.m. injection in mice. At the lower dose tested (i.e., 3x10 ¹⁰ GC/mouse), the difference in AAV8triple expression is significant.

Example 9. Expression of RAAV vector transgene in male C57BL/6J mice.

Expression in liver and muscle was tested in vivo using rAAV vectors with different capsids, including AAVhu68, AAV8triple, AAV8, and AAV9, and expressing firefly luciferase (ffLuc) as a transgene.

Male C57BL/6J mice aged 6–8 weeks (n=5/group) were injected intramuscularly into the gastrocnemius muscle with 3 x 1011 GC/mouse of the test vector using a Hamilton syringe. ffLuc expression was visualized by weekly whole-body bioluminescence imaging as described

- 55 048431 previously (Greig et al., PLoS One 2014, cited above).

As shown in Fig. 11A and 11B, AAVhu68, AAV8, and AAV9 vectors were expressed at similar levels in both muscle and liver, while AAV8triple vector had decreased expression in liver and increased expression in muscle.

Example 10. RAAV vectors in male and female CYNOMOLGUS macaques.

Transgene expression was tested in cynomolgus macaques using rAAV vectors having different capsids, including AAVhu68, AAV8triple, AAV8, and AAV9, and expressing the secreted transgene, 201Ig IA.

To study vector biodistribution, male and female cynomolgus macaques with nAb titers to the injected vector <1:5 at baseline were injected intramuscularly into the vastus lateralis muscle of the right and left legs at a dose of 10 GC/kg body weight with a vector expressing 201Ig IA with one of four vector capsids (AAV8triple, AAVhu68, AAV9, or AAV8) as 1 ml/kg body weight injections (vector concentration 10 ¹³ GC/ml). Blood samples were collected before the study and weekly during the study by venipuncture of the femoral vein. Transgene expression levels were measured in serum by ELISA as described previously (Greig et al., PLoS One 2014, cited above).

As shown in Fig. 12, after i.m. injection, AAVhu68 and AAV8triple vectors were expressed better than AAV9 and AAV8.

All documents cited in this specification are incorporated herein by reference, as are U.S. Provisional Patent Application No. 62/614,002, filed January 5, 2018, U.S. Provisional Patent Application No. 62/591,001, filed November 27, 2017, and U.S. Provisional Patent Application No. 62/464,748, filed February 28, 2017. The sequence listing filed with this document, designated 17-7986 Seq Listing_ST25.txt, and the sequences and text therefor, are incorporated herein by reference. The invention has been described with reference to specific embodiments thereof, but it is to be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be within the scope of the appended claims.

Arbitrary text of a sequence list.

The following information is provided for sequences containing arbitrary text under the numeric identifier <223>.

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SEQ GO NO: (contains arbitrary text) Custom text under <223> 2 <223> Synthetic construction 3 <223> Gene rep AAVhu68 of Homo Sapiens origin 4 <223> Synthetic construction 5 <223> VP1 AAV9 capsid of Homo Sapiens origin <220> <221> CDS <222> (1)..(2208) <223> VP1 AAV9 capsid 6 <223> Synthetic construction 7 <223> Primer prm504 8 <223> Primer prm505 9 <223> AAVhu68 spacer sequence 10 <223> Capsid protein vpl AAVhu31 11 <223> Capsid protein vpl AAVhu32 12 <223> Coding sequence vpl AAVhu31 13 <223> Coding sequence vpl AAVhu32 14 <223> modified hu68vpl <220> <221> OTHER TRAIT <222> (23)..(23) <223> Xaa can be W (Tgr, tryptophan) or

- 57 048431

oxidized W. <220> <221> OTHER CHARACTER <222> (35)..(35) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER CHARACTER <222> (57)..(57) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER CHARACTER <222> (66)..(66) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER CHARACTER <222> (94)..(94) <223> Xaa may be Asn or deamidated to Asp, iso Asp or Asp/isoAsp <220> <221> OTHER CHARACTER <222> (97)..(97) <223> Xaa may be D (asp, aspartic acid) or isomerized D. <220> <221> OTHER CHARACTER <222> (107)..(107) <223> Xaa may be D (asp, aspartic acid) or isomerized D. <220> <221> other character <222> (113)..(113) <223> Xaa may be any amino acid

- 58 048431

naturally occurring <220> <221> OTHER TRAIT <222> (149)..(149) <223> Xaa may be S (Ser, serine) or phosphorylated S <220> <221> OTHER TRAIT <222> (149)..(149) <223> Xaa may be S (Ser, serine) or phosphorylated S <220> <221> OTHER TRAIT <222> (247)..(247) <223> Xaa may be W (Trp, tryptophan) or oxidized W (e.g., kynurenine). <220> <221> OTHER FAULT <222> (253)..(253) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER FAULT <222> (259)..(259) <223> Xaa is Q, or Q deamidated to glutamic acid (alpha-glutamic acid), gamma-glutamic acid (Glu), or a mixture of alpha- and gamma-glutamic acids <220> <221> OTHER FAULT <222> (270)..(270) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220>

- 59 048431

<221> OTHER TRAIT <222> (297)..(297) <223> Xaa is D (Asp, aspartic acid) or amidated with D to N (Asn, asparagine) <220> <221> OTHER TRAIT <222> (304)..(304) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER TRAIT <222> (306)..(306) <223> Xaa may be W (Trp, tryptophan) or oxidized W (e.g., kynurenine). <220> <221> OTHER CHARACTER <222> (314)..(314) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER CHARACTER <222> (319)..(319) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER CHARACTER <222> (329)..(329) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER CHARACTER <222> (332)..(332) <223> Xaa can be K (lys, lysine) or acetylated K

- 60 048431

<220> <221> OTHER FAULT <222> (336)..(336) <223> Xaa may be Asn or deamidated to Asp, iso Asp or Asp/isoAsp <220> <221> OTHER FAULT <222> (384)..(384) <223> Xaa may be D (asp, aspartic acid) or isomerized D. <220> <221> OTHER FAULT <222> (404)..(404) <223> Xaa may be M (Met, methionine) or oxidized M. <220> <221> OTHER FAULT <222> (409)..(409) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER INDICATION <222> (436)..(436) <223> Xaa may be M (Met, methionine) or oxidized M. <220> <221> OTHER INDICATION <222> (452)..(452) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER INDICATION <222> (477)..(477) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp

- 61 048431

<220> <221> OTHER FAULT <222> (499)..(499) <223> Xaa may be S (Ser, serine) or phosphorylated S <220> <221> OTHER FAULT <222> (512)..(512) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER FAULT <222> (515)..(515) <223> Xaa may be Asn or deamidated to Asp, iso Asp, or Asp/isoAsp <220> <221> OTHER FAULT <222> (518)..(518) <223> Xaa may be M (Met, methionine) or oxidized M. <220> <221> OTHER TRAIT <222> (524)..(524) <223> Xaa may be M (Met, methionine) or oxidized M. <220> <221> OTHER TRAIT <222> (559)..(559) <223> Xaa may be M (Met, methionine) or oxidized M. <220> <221> OTHER TRAIT <222> (569)..(569) <223> Xaa may be T (Thr, threonine) or phosphorylated T

- 62 048431

<220> <221> OTHER TRAIT <222> (586)..(586) <223> Xaa may be S (Ser, serine) or phosphorylated S <220> <221> OTHER TRAIT <222> (599)..(599) <223> Xaa is Q, or Q deamidated to glutamic acid (alpha-glutamic acid), gamma-glutamic acid (Glu), or a mixture of alpha- and gamma-glutamic acids <220> <221> OTHER TRAIT <222> (605)..(605) <223> Xaa may be M (Met, methionine) or oxidized M. <220> <221> OTHER TRAIT <222> (619)..(619) <223> Xaa may be W (Trp, tryptophan) or oxidized W (e.g., kynurenine). <220> <221> OTHER TRAIT <222> (628)..(628) <223> Xaa may be Asn or deamidated to Asp, isoAsp, or Asp/isoAsp <220> <221> OTHER TRAIT <222> (640)..(640) <223> Xaa may be M (Met, methionine) or oxidized M. <220> <221> OTHER TRAIT <222> (651)..(651)

- 63 048431

<223> Haa may represent Asn or be deamidated to Asp, iso Asp or Asp/isoAsp <220> <221> OTHER CHARACTERISTIC <222> (663)..(663) <223> Xaa may be deamidated to Asp, iso Asp or Asp/isoAsp <220> <221> OTHER CHARACTERISTIC <222> (666)..(666) Asn or be <223> Xaa may represent K acetylated K <220> <221> OTHER SIGN <222> (689)..(689) (lys, lysine) or <223> Xaa may represent K acetylated K <220> <221> OTHER SIGN <222> (693)..(693) (lys, lysine) or <223> Xaa may represent K acetylated K <220> <221> OTHER SIGN <222> (695)..(695) (lys, lysine) or

<223> Xaa may be W (Trp, tryptophan) or oxidized W.

<220>

<221> OTHER SIGN <222> (709)..(709)

<223> Xaa may be deamidated to Asp, iso Asp, or <220> <221> OTHER CHARACTERISTIC <222> (735)..(735) by yourself Asp/isoAsp Asn or be <223> Xaa can be presented deamidated to Asp, iso Asp or by yourself Asp/isoAsp Asn or be

Claims (10)

CLAUSE OF THE INVENTION 1. A recombinant adeno-associated virus (rAAV) that comprises a vector genome in an AAVhu68 capsid, wherein (A) the vector genome comprises a nucleic acid molecule comprising AAV inverted terminal repeat (ITR) sequences and, between the ITRs, a non-AAV nucleic acid sequence encoding a product operably linked to sequences that direct expression of that product in a mammalian target cell; and (B) the AAVhu68 capsid comprises AAVhu68 vp1 proteins, AAVhu68 vp2 proteins and AAVhu68 vp3 proteins derived from a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, wherein the AAVhu68 vp1 proteins comprise a glutamic acid at position 67 and a valine at position 157, and the AAVhu68 vp2 proteins comprise a valine at position 157 based on the numbering of SEQ ID NO: 2. 2. The rAAV of claim 1, wherein the AAVhu68 comprises subpopulations of the AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 vp3 proteins, wherein the subpopulations of the AAVhu68 vp1, AAVhu68 vp2, and AAV hu68 vp3 proteins comprise at least 50 to 100% deamidated asparagines (N) in asparagine-glycine pairs at each of positions 57, 329, 452, and 512 relative to the amino acids of SEQ ID NO: 2, wherein the deamidated asparagines are deamidated to aspartic acid, isoaspartic acid, an aspartic acid/isoaspartic acid interconvertible pair, or combinations thereof, as determined by mass spectrometry. 3. A recombinant adeno-associated virus (rAAV) that contains the AAVhu68 capsid and the vector genome in the AAVhu68 capsid, where: (A) the vector genome comprises a nucleic acid molecule comprising AAV inverted terminal repeat (ITR) sequences and, between the ITRs, a non-AAV nucleic acid sequence encoding a product operably linked to sequences that direct expression of the product in a mammalian target cell; and (B) the AAVhu68 capsid comprises heterogeneous populations of AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 vp3 proteins, wherein the AAVhu68 vp1 proteins are amino acids 1 to 736 of SEQ ID NO: 2 (vpl) that contain a glutamic acid at position 67 and a valine at position 157 and further comprise subpopulations of vp1 proteins comprising modified amino acids based on the amino acid positions of SEQ ID NO: 2, wherein the AAVhu68 vp2 proteins are amino acids 138 to 736 of SEQ ID NO: 2 (vp2) that contain a valine at position 157 and further comprise subpopulations of vp2 proteins comprising modified amino acids based on the amino acid positions of SEQ ID NO: 2, and wherein the AAVhu68 vp3 proteins are amino acids 203 to 736 of SEQ ID NO: 2 (vp3), which comprise subpopulations of vp3 proteins comprising modified amino acids based on the amino acid positions of SEQ ID NO: 2, wherein the subpopulations of AAVhu68 vp1, AAVhu68 vp2, and AAV hu68 vp3 proteins comprise at least 50 to 100% deamidated asparagines (N) in asparagine-glycine pairs at each of positions 57, 329, 452, and 512 relative to the amino acids of SEQ ID NO: 2, wherein the deamidated asparagines are deaminated to aspartic acid, isoaspartic acid, an aspartic acid/isoaspartic acid interconvertible pair, or combinations thereof, as determined by mass spectrometry. 4. The rAAV of claim 2 or 3, wherein the vp1, vp2 and vp3 protein subsets of AAVhu68 further comprise (i) one or more additional modifications selected from acetylated lysine, phosphorylated serine and/or threonine, isomerized aspartic acid, deamidated glutamines, oxidized tryptophan and/or methionine, or an amidated amino acid, as determined by mass spectrometry; and/or (ii) from 1 to 40% deamidation of asparagines at one or more positions N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N336, N409, N410, N477, N515, N598, Q599, N628, N651, N663, N709 or a combination thereof, based on the numbering of SEQ ID NO: 2, determined by mass spectrometry. 5. The rAAV of any one of claims 2 to 4, wherein the AAVhu68 capsid subpopulations of the AAVhu68 capsid proteins vp1, vp2, and vp3 further comprise (a) at least 65% of the asparagines (N) in the asparagine-glycine pairs located at positions 57 of the vp1 proteins are deamidated, as determined by mass spectrometry based on the residue numbering of SEQ ID NO: 2; and/or (b) at least 75% of the N in the asparagine-glycine pairs at position 329 of the vp1, v2, and vp3 proteins are deamidated, as determined by mass spectrometry based on the residue numbering of the amino acid sequence of SEQ ID NO: 2; and/or - 65 048431 (c) at least 50% of the N in the asparagine-glycine pairs at position 452 of the proteins vp1, v2 and vp3 are deamidated, as determined by mass spectrometry, based on the residue numbering of the amino acid sequence of SEQ ID NO: 2; and/or (d) at least 75% of the N in the asparagine-glycine pairs at position 512 of the proteins vp1, vp2 and vp3 are deamidated, as determined by mass spectrometry, based on the residue numbering of the amino acid sequence of SEQ ID NO: 2. 6. The rAAV of any one of claims 1 to 5, wherein the AAVhu68 capsid comprises: (a) a subpopulation of vp1 proteins in which between 75 and 100% of the N at position 57 of the vp1 proteins is deamidated, as determined by mass spectrometry; and/or (b) a subpopulation of vp1 proteins, vp2 proteins and/or vp3 proteins in which between 75 and 100% of the N at position 329, based on the numbering of SEQ ID NO: 2, is deamidated, as determined using mass spectrometry; and/or (c) a subpopulation of vp1 proteins, vp2 proteins and/or vp3 proteins in which between 75 and 100% of the N at position 452, based on the numbering of SEQ ID NO: 2, is deamidated, as determined using mass spectrometry; and/or (d) a subset of vp1 proteins, vp2 proteins and/or vp3 proteins in which between 75 and 100% of the N at position 512 based on the numbering of SEQ ID NO: 2 is deamidated, as determined using mass spectrometry. 7. The rAAV of any one of claims 1 to 6, wherein the nucleic acid sequence encoding the proteins is SEQ ID NO: 1, or a sequence at least 80% to at least 99% identical to the sequence of SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2; optionally, the nucleic acid sequence is at least 80-97% identical to SEQ ID NO: 1. 8. A composition for delivering a desired gene product to a subject in need thereof, comprising a mixed population of recombinant adeno-associated virus hu68 (rAAVhu68), wherein each rAAVhu68 is independently selected from the rAAV of any one of claims 1-7. 9. The rAAV of any one of claims 1 to 7 or the composition of claim 8, wherein the AAV ITR sequences are the 5' ITR and 3' ITR of AAV2. 10. The composition of claim 8 or 9, wherein the composition is formulated for (i) intrathecal delivery and the vector genome comprises a nucleic acid sequence encoding a product for delivery to the central nervous system; (ii) intravenous delivery; or (iii) intranasal or intramuscular delivery.