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WO2016196366A1 - Extension de durée de vie réplicative dans des maladies de vieillissement prématuré à l'aide d'isoformes p53 - Google Patents

Extension de durée de vie réplicative dans des maladies de vieillissement prématuré à l'aide d'isoformes p53 Download PDF

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Publication number
WO2016196366A1
WO2016196366A1 PCT/US2016/034821 US2016034821W WO2016196366A1 WO 2016196366 A1 WO2016196366 A1 WO 2016196366A1 US 2016034821 W US2016034821 W US 2016034821W WO 2016196366 A1 WO2016196366 A1 WO 2016196366A1
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cell
expression
cells
disease
premature aging
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PCT/US2016/034821
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English (en)
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Curtis C. Harris
Izumi Horikawa
Natalia VON MUHLINEN
Casmir TURNQUIST
David Lane
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The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
Agency For Science, Technology And Research
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Publication of WO2016196366A1 publication Critical patent/WO2016196366A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Replicative senescence a state of irreversible growth arrest, can be triggered by multiple mechanisms including telomere shortening, oncogene activation, and DNA damage. These mechanisms limit excessive or aberrant cellular proliferation and put senescence in the crossroads of protecting individuals from cancer progression and contributing to organismal aging.
  • p53 is a protein of having an apparent molecular weight of about 53 kDa on SDS PAGE that functions as a transcription factor that, among other functions, regulates the cell cycle and acts as a tumor suppressor. In normal cells, p53 is generally held in an inactive form, bound to the protein MDM2 (HDM2 in humans), which prevents p53 activity and promotes p53 degradation by acting as a ubiquitin ligase. Active p53 is induced in response to various cancer-causing agents such as UV radiation, oncogenes, and some DNA-damaging drugs.
  • DNA damage is sensed by 'checkpoints' in a cell's cycle, and causes proteins such as ATM, CHK1 and CHK2 to phosphorylate p53 at sites that are close to or within the MDM2- binding region and p300-binding region of the protein.
  • Oncogenes also stimulate p53 activation, mediated by the protein p14ARF. Some oncogenes can also stimulate the transcription of proteins which bind to MDM2 and inhibit its activity. Once activated, p53 activates expression of several genes including one encoding for p21, a cell cycle inhibitor. p21 binds to G1-S-phase and S-phase cyclin CDK complexes inhibiting their activity. See, e.g., Mills, Genes & Development, 19: 2091-2099 (2005) for a review.
  • the p53 signaling network plays an important role in regulating senescence.
  • the p53 gene tumor protein p53 gene TP53 encodes at least 13 natural isoforms due to alternative splicing and alternative promoter usage. It has previously been shown that a natural isoform of p53, ⁇ 133p53, preferentially inhibits p53- target genes involved in cellular senescence in normal cells (see, e.g., WO 2009/064590).
  • WS Werner syndrome
  • HGPS Hutchinson-Gilford progeria syndrome
  • WS is an autosomal syndrome caused by mutations in the DNA-repair essential WRN helicase gene that result in premature aging in young adults.
  • HGPS is a laminopathy caused by mutations in the LMNA gene resulting in post-translational processing defects that lead to an abnormal nuclear envelope structure and causes Progeria in children.
  • Fibroblasts derived from individuals with WS or HGPS have decreased cellular proliferation and increased senescence in culture, as well as altered DNA damage responses.
  • the invention provides a method of extending the replicative lifespan of a cell of a subject that has a disease of premature aging, the method comprising contacting the cell with an agent that increases the function or expression of ⁇ 133p53, thereby inhibiting cell senescence and extending the replicative lifespan of the cell.
  • the disease of premature aging is Werner syndrome or Hutchinson-Gilford progeria syndrome.
  • the agent comprises a polynucleotide sequence encoding ⁇ 133p53, e.g., the agent may comprise an expression cassette comprising a polynucleotide sequence encoding ⁇ 133p53.
  • the cell is a stem cell.
  • the cell is a cardiac progenitor cell, an endothelial progenitor cell, or a bone marrow-derived progenitor cell.
  • the cell is contacted with the agent ex vivo. In some embodiments, the cells are then expanded and administered to the patient.
  • the invention provides a method of extending the replicative lifespan of a cell of a subject that has a disease of premature aging, the method comprising contacting the cell with an agent that inhibits the function or expression of miR-34a, thereby inhibiting cell senescence and extending the replicative lifespan of the cell.
  • the disease of premature aging may be Werner syndrome or Hutchinson- Gilford progeria syndrome.
  • the agent comprises a polynucleotide sequence that inhibits miR-34a, e.g., an expression cassette comprising a polynucleotide sequence that inhibits miR-34a.
  • the cell is a stem cell.
  • the cell is a cardiac progenitor cell, an endothelial progenitor cell, or a bone marrow-derived progenitor cell. In some embodiments, the cell is contacted with the agent ex vivo. In some embodiments, the cells are then expanded and administered to the patient.
  • the invention provides a method of treating a disease of premature aging, the method comprising administering a cell modified in accordance with any method described herein to increase ⁇ 133p53 expression or activity, or to inhibit miR- 34a activity, to a patient that has a disease of premature aging.
  • the disease of premature aging is Werner syndrome or Hutchinson-Gilford progeria syndrome.
  • the invention provides a method of extending the replicative lifespan of a cell of a subject that has a disease of premature aging, the method comprising contacting the cell with an agent that inhibits the function or expression of p53 ⁇ , thereby inhibiting cell senescence and extending the replicative lifespan of the cell.
  • the disease of premature aging is Werner syndrome.
  • the disease of premature aging is Hutchinson-Gilford progeria syndrome.
  • the agent comprises a polynucleotide sequence that inhibits p53 ⁇ , or comprises an expression cassette comprising a polynucleotide sequence that inhibits p53 ⁇ .
  • the cell is a stem cell.
  • cell is a cardiac progenitor cell or an endothelial progenitor cell. In some embodiments, the cell is a bone marrow-derived progenitor cell.
  • the cell may be contacted with the agent in vivo. In some embodiments, the cell is contacted with the agent ex vivo.
  • the invention thus provides a method of treating a disease of premature aging, the method comprising administering a cell modified ex vivo to increase expression of ⁇ 133p53 or decrease expression of p53 ⁇ as described in herein to a patient that has a disease of premature aging.
  • the disease of premature aging may be Werner syndrome or Hutchinson-Gilford progeria syndrome.
  • FIG. 1A-1D provides illustrative data showing that ⁇ 133p53 protects Werner syndrome fibroblasts (AG00780) from senescence and extends their replicative lifespan.
  • SASP senescence-associated secretory phenotype
  • Figure 2A-2D provides illustrative data showing that ⁇ 133p53 protects WS fibroblasts (AG03141) from senescence and extends their replicative lifespan.
  • Figure 4A-4B provides illustrative data showing that expression of ⁇ 133p53 stabilizes full-length p53 (FL-p53), but inhibits the senescent-associated p53-target gene p21 WAF1 in WS fibroblasts.
  • Expression of ⁇ 133p5 in cells derived from WS patients (AG00780, AG03141 and AG12797) resulted in (4A) accumulated full-length p53 (FL-p53) at the protein level and (4B) inhibition of p21 WAF 1 expression at the mRNA level.
  • Data are mean ⁇ s.d. from three independent experiments. *** P ⁇ 0.001
  • FIG. 5A-5B provides illustrative data showing that ⁇ 133p53 is downregulated during passage of Hutchinson-Gilford Progeria Syndrome (HGPS) fibroblasts (AG11513).
  • HGPS Hutchinson-Gilford Progeria Syndrome
  • 5A AG11513 proliferated for a limited number of population doublings (PDLs) in culture before they reached growth arrest.
  • 5B ⁇ 133p53 protein levels were downregulated in late- passage (passage 14) compared to early-passage (passage 7) AG11513 fibroblasts derived from a patient with Hutchinson-Gilford Progeria Syndrome (HGPS).
  • FIG. 6A-6D provides illustrative data showing that ⁇ 133p53 protects HGPS fibroblasts (AG11513) from senescence and extends their replicative lifespan.
  • Figure 7A-7D provides illustrative data showing that ⁇ 133p53 protects HGPS fibroblasts (AG01972) from senescence and extends their replicative lifespan.
  • 7A Expression of ⁇ 133p53 isoform in AG01972 derived from a patient with Hutchinson-Gilford Progeria Syndrome (HGPS) resulted in: (7B) increased lifespan; and (7C, 7D) diminished premature senescence as shown by (7C) decreased SA- ⁇ -gal staining and (7D) lower expression of IL-6, a senescence-associated secretory phenotype (SASP) cytokine.
  • SASP senescence-associated secretory phenotype
  • Figure 8A-8C provides illustrative data showing that ⁇ 133p53 dominant-negatively inhibits the senescence-associated p53 targets p21 WAF1 and miR-34a, and stabilizes full- length p53 (FL-p53) and phosphorylated p53 at serine 15 (pS15-p53) in AG11513 (HGPS) fibroblasts.
  • Figure 9A-9B provides illustrative data showing that expression of the DNA-repair gene RAD51 is upregulated in ⁇ 133p53-expressing HGPS cells.
  • Expression of ⁇ 133p53 in HGPS fibroblasts resulted in increased expression of the DNA repair factor RAD51 (9A) at the mRNA level as shown by quantitative RT-PCR and (9B) at the protein level as shown by western blot.
  • 9A control, left bar in each set; ⁇ 133p53, right bar in each set.
  • Data are mean ⁇ s d from three independent experiments.
  • Figure 10A-10C provides illustrative data showing that expression of ⁇ 133p53 ameliorates spontaneous DNA damage by increasing recruitment of RAD51 to ⁇ H2AX- positive foci in HGPS fibroblasts.
  • Cells were co-immunostained with antibodies against ⁇ H2AX and RAD51.
  • (10A) Representative images are shown. Scale bar 10 ⁇ m.
  • ⁇ H2AX- positive foci were counted and co-localization with RAD51was scored as RAD51-positive (RAD51+ve) or RAD51-negative (RAD51-ve). Results are shown as percentage (%) of ⁇ H2AX-positive foci. The area inside the columns represents RAD51-positive foci (grey) or RAD51-negative (white). Data are mean ⁇ s.d. from three independent experiments. ** P ⁇ 0.01.
  • FIG. 11 A-11D provide illustrative data showing that p53 ⁇ expression is associated with senescence of HGPS fibroblasts.
  • 11C, 11D Overexpression of p53 ⁇ or a control vector in proliferative HGPS fibroblasts accelerated the onset of senescence in HGPS fibroblasts.
  • FIG. 12 A-12E provides illustrative data showing that SRSF3 regulates p53 ⁇ expression during senescence of HGPS.
  • p53 refers generally to a protein of a molecular weight of about 53kDa on SDS PAGE that functions as a transcription factor and plays a role in cell cycle control and DNA damage repair as well as other essential pathways in cell growth.
  • the protein and nucleic sequences of the p53 protein from a variety of organisms from humans to Drosophila are known and are available in public databases, such as in accession numbers, NM_000546, NP_000537, NM_011640, and NP_035770, for the human and mouse sequences.
  • Mammalian p53 sequences are highly conserved between species. Mouse and human p53 proteins are 85% identical. In humans, p53 is encoded by the TP53 gene located on the short arm of chromosome 17 (17p13.1).
  • a p53 protein in the context of this invention includes allelic variants and other functional variants and orthologs. In some embodiments, variants have at least 85%, at least 90%, or at least 95%, or greater, amino acid sequence identity across their whole sequence compared to a naturally occurring p53 family member, such as the polypeptide sequence listed under accession number NP_000537 (human p53).
  • ⁇ 133p53 refers generally to the isoform of p53 that arises from initiation of transcription of the p53 gene from an alternative promoter in intron 4 of human p53, which results in an N-terminally truncated p53 protein translated from codon 133.
  • a ⁇ 133p53 isoform for use in this invention comprises the following p53 protein domains: the majority of the DNA binding domain, the NLS, and the C-terminal oligomerization domain (see Bourdon, Brit. J. Cancer, 97: 277-282 (2007)).
  • An illustrative human ⁇ 133p53 protein sequence is listed under accession number ABA29755.1.
  • p53 ⁇ refers generally to the isoform of p53 that arises from alternative splicing of intron 9 to provide a p53 isoform comprising the following p53 protein domains: TAD1, TAD2, prD, the DNA binding domain, the NLS, and the C-terminal sequence DQTSFQKENC (see Bourdon, Brit. J. Cancer, 97: 277-282 (2007)).
  • cell senescence refers generally to the phenomenon where normal diploid differentiated cells lose the ability to divide after undergoing a finite number of cell divisions characteristic of a particular type of cell.
  • replicaative lifespan refers generally to the finite number of cell divisions undergone by a particular cell type before undergoing cell senescence and losing the ability to further divide.
  • the term“extending replicative lifespan” refers generally to the continuation of cell division in a normal diploid cell beyond the finite number of cell divisions at which cell senescence would occur.
  • inhibiting refers generally to conditions or agents which reduce, decrease, hinder, down- regulate, or otherwise decrease cell senescence compared to a corresponding cell that is not treated with the agent.
  • the term“expression” or“increasing expression” of ⁇ 133p53 in the context of this invention often refers to introducing a ⁇ 133p53 nucleic acid into a cell.
  • the level of expression of ⁇ 133p53 may be increased using another agent that upregulates expression.
  • An "increase" in ⁇ 133p53 expression is generally determined relative to control cells to which an agent, e.g., a nucleic acid encoding ⁇ 133p53 or other agent that increases levels of ⁇ 133p53, has not been added.
  • Expression is typically considered to be increased when levels of ⁇ 133p53 protein, or RNA, are increased by at least 20%, typically by at least 50%, or 100% or more.
  • expression levels may be two-fold higher than the control, or five-fold higher than the control, or greater than five-fold higher than the control.
  • Expression of ⁇ 133p53 can also be evaluated by functional analyses, such as measuring cellular senescence, e.g., using a Senescence- Associated (SA)- ⁇ -Galactosidase assay to determine cellular senescence levels, or evaluating expression of IL- 6, a senescence-associated secretory phenotype (SASP) cytokine.
  • SA Senescence- Associated
  • SASP senescence-associated secretory phenotype
  • the term“decreasing” or“inhibiting” expression of p53 ⁇ in the context of this invention refers to decreasing the level of p53 ⁇ or activity in a cell.
  • the level of expression of p53 ⁇ may be decreased using a polynucleotide inhibitor of p53 ⁇ .
  • another agent that down regulates expression is employed.
  • a "decrease" in p53 ⁇ expression is generally determined relative to control cells to which an agent, e.g., a nucleic acid encoding a polynucleotide inhibitor of p53 ⁇ , has not been added.
  • Expression is typically considered to be decreased when levels of p53 ⁇ protein or RNA, are decreased by at least 10% or at least 20%, typically by at least 50%, or 75% or more. In some embodiments, expression levels may be two-fold lower than the control, or five-fold lower than the control, or greater than five-fold lower than the control. As noted above, in some embodiments, inhibited expression of p53 ⁇ can be assessed using functional analyses, such as measuring cellular senescence, e.g., using a Senescence- Associated (SA)- ⁇ -Galactosidase assay to determine cellular senescence levels, or evaluating expression of IL- 6, a senescence-associated secretory phenotype (SASP) cytokine.
  • SA Senescence- Associated
  • SASP senescence-associated secretory phenotype
  • a polynucleotide sequence is“heterologous to” a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form.
  • a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants.
  • exogenous refers to a substance present in a cell or organism other than its native source.
  • exogenous nucleic acid or “exogenous protein” refer to a nucleic acid or protein that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found or in which it is found in lower amounts.
  • a substance will be considered exogenous if it is introduced into a cell or an ancestor of the cell that inherits the substance.
  • endogenous refers to a substance that is native to the biological system.
  • identity refers to the extent to which the sequence of two or more nucleic acids or polypeptides is the same.
  • the percent identity between a sequence of interest and a second sequence over a window of evaluation may be computed by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the window of evaluation that are opposite an identical residue allowing the introduction of gaps to maximize identity, dividing by the total number of residues of the sequence of interest or the second sequence (whichever is greater) that fall within the window, and multiplying by 100.
  • fractions are to be rounded to the nearest whole number.
  • Percent identity can be calculated with the use of a variety of computer programs. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments and provide percent identity between sequences of interest.
  • the algorithm of Karlin and Altschul Karlin and Altschul, Proc. Natl. Acad. ScL USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc. Natl. Acad. ScL USA 90:5873-5877, 1993 is incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. MoI. Biol.215:403-410, 1990).
  • Gapped BLAST is utilized as described in Altschul et al. (Altschul, et al. Nucleic Acids Res.25: 3389-3402, 1997).
  • the default parameters of the respective programs may be used.
  • a PAM250 or BLOSUM62 matrix may be used.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). See the Web site having URL world-wide web address of: "ncbi.nlm.nih.gov" for these programs.
  • percent identity is calculated using BLAST2 with default parameters as provided by the NCBI.
  • isolated refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and/or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides.
  • a chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation is considered “isolated”.
  • isolated cell refers to a cell that has been removed from an organism in which it was originally found or a descendant of such a cell.
  • An“isolated” cell may be cultured in vitro in the presence of other cells.
  • the cell is later introduced into a second organism or re-introduced into the organism from which it (or the cell from which it is descended) was isolated.
  • vector refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell.
  • vectors of use in the invention are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors".
  • an "expression vector” is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell.
  • the gene of interest is operably linked to another sequence in the vector, e.g., a promoter.
  • Vectors include non-viral vectors such as plasmids and viral vectors.
  • operably linked refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence, such that the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Operably linked nucleic acid sequences need not be physically adjacent to each other.
  • the term“operably linked” also refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a transcribable nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the transcribable sequence.
  • viral vectors refers to the use of viruses, or virus-associated vectors as carriers of a nucleic acid construct into a cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral and lentiviral vectors, for infection or transduction into cells.
  • AAV Adeno-associated virus
  • HSV Herpes simplex virus
  • the vector may or may not be incorporated into the cell's genome.
  • the constructs may include viral sequences for transfection, if desired.
  • the construct may be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors.
  • transfection is used to refer to any method of introducing nucleic acid molecules into a cell including both viral and non-viral techniques.
  • the term as used here thus includes techniques such as transduction with a virus.
  • regulatory sequence and “promoter” are used interchangeably herein, and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operatively linked.
  • transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell- type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of a protein.
  • the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required for initiating transcription of a specific gene.
  • proliferating and proliferation refer to an increase in the number of cells in a population (growth) by means of cell division.
  • Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, including growth factors and other mitogens.
  • Cell proliferation may also be promoted by release from the actions of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.
  • nucleic acid and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.
  • “Conservatively modified variants” as used herein applies to amino acid sequences.
  • One of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • the disclosure is based, in part on the discovery that the naturally occurring p53 isoform ⁇ 133p53 extends the replicative lifespan of cells in patients who have diseases of premature aging.
  • Premature aging disease or conditions are those in which aspects of the aging process, e.g., external signs of old age, is greatly accelerated.
  • Children affected with a disease of premature aging develop external signs of old age, including baldness, hunched posture, and dry, inelastic, and wrinkled skin.
  • the ovaries or testes are inactive, resulting in sterility. Affected children are also unusually short.
  • the invention thus provides methods of extending replicative life-span in cells of patients that have a pre-mature aging syndrome, e.g., WS or HGPS, by increasing the level or activity of ⁇ 133p53 in cells of such patients.
  • Increasing the level or activity of ⁇ 133p53 may be achieved using any number of methods, including, but not limited to, introducing a polynucleotide encoding ⁇ 133p53 into cells, e.g., an expression vector that expresses ⁇ 133p53, and/or using an agent that upregulates ⁇ 133p53 expression.
  • cells from a patient may be modified ex vivo and then re- introduced into the patient.
  • cells that are modified ex vivo for reintroduction into the patient are stem cells or progenitor cells, such as cardiac progenitor cells, bone marrow-derived stem cells, or endothelial progenitor cells.
  • the patient may be treated with an agent that up-regulates ⁇ 133p53 expression and/or activity.
  • an agent targets a cellular autophagy degradation pathway.
  • the invention provides methods of identifying agents that increase ⁇ 133p53 levels and/or activity in cells of patients having a disease of pre-mature aging, e.g., WS or HGPS, where the method comprises screening candidate small molecule compounds for the ability up-regulate ⁇ 133p53 expression or activity in WS and/or HGPS cells.
  • the disclosure is additionally based, in part on the discovery that expression of the naturally occurring p53 isoform p53 ⁇ is associated with senescence in cells of patients who have a premature aging disease.
  • the disclosure thus provides methods of extending replicative life-span in cells of patients that have a pre-mature aging syndrome, e.g., WS or HGPS, by decreasing the level or activity of p53 ⁇ in cells of such patients. Decreasing the level or activity of p53 ⁇ may be achieved using any number of methods, including, but not limited to, introducing a polynucleotide that inhibits expression of p53 ⁇ into cells and/or using an agent that downregulates p53 ⁇ expression.
  • cells from a patient may be modified ex vivo to inhibit expression of p53 ⁇ and then re-introduced into the patient.
  • cells that are modified ex vivo for reintroduction into the patient are stem cells or progenitor cells, such as cardiac progenitor cells, bone marrow-derived stem cells, or endothelial progenitor cells.
  • the patient may be treated with an agent that down-regulates p53 ⁇ expression and/or activity.
  • the invention provides methods of identifying agents that decrease p53 ⁇ levels and/or activity in cells of patients having a disease of pre-mature aging, e.g., WS or HGPS, where the method comprises screening candidate small molecule compounds for the ability down-regulate p53 ⁇ expression or activity in WS and/or HGPS cells.
  • Nucleic acid sequences encoding ⁇ 133p53 and related nucleic acid sequence homologues can be cloned from cDNA libraries or are typically isolated using amplification techniques with oligonucleotide primers.
  • the cloning of ⁇ 133p53, or other p53 isoforms, or desired genes can employ the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the nucleic acids encoding ⁇ 133p53 are typically cloned into intermediate vectors before transformation into eukaryotic cells for replication and/or expression. These intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors.
  • the isolated nucleic acids encoding ⁇ 133p53 or other p53 isoforms may also encode interspecies homologues, alleles and polymorphic variants of the illustrative ⁇ 133p53 described herein.
  • ⁇ 133p53 (or of other proteins that it may be desirable to express as described herein) is performed using a variety of techniques.
  • Basic texts disclosing general methods that can be employed in this invention include Green and Sambrook (2012) Molecular Cloning: A laboratory manual 4th ed. Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology and supplements through supplement 110, 2015) John Wiley and Sons.
  • a nucleic acid construct encoding ⁇ 133p53 is a plasmid- based vector (e.g.,“naked” DNA).
  • a nucleic acid construct encoding ⁇ 133p53 is contained within a viral vector and administered as a virus.
  • Viral delivery systems include adenovirus vectors, adeno-associated viral vectors, herpes simplex viral vectors, retroviral vectors, pox viral vectors, lentiviral vectors, alphavirus vectors, poliovirus vectors, and other positive and negative stranded RNA viruses, viroids, and virusoids, or portions thereof.
  • recombinant viruses in the pox family of viruses can be used for delivering the nucleic acid molecules encoding ⁇ 133p53.
  • vaccinia viruses include vaccinia viruses and avian poxviruses, such as the fowlpox and canarypox viruses.
  • Illustrative methods for generating these viruses using genetic recombination can be found, e.g, in WO 91/12882; WO 89/03429; WO 92/03545; and n US Patent No.5,863,542.
  • Representative examples of recombinant pox viruses include ALVAC, TROVAC, and NYVAC.
  • a number of adenovirus vectors can also be used to deliver a nucleic acid that encodes ⁇ 133p53 (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K. L.
  • AAV vector systems have been developed for gene delivery. These vectors can be readily constructed. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan.1992) and WO 93/03769 (published 4 Mar.1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.
  • Retroviruses also provide a platform for gene delivery systems.
  • a number of retroviral systems have been described (U.S. Pat. No.5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.
  • Additional gene delivery systems include lentiviral vectors that employ lentiviral vector backbones.
  • a nucleic acid encoding ⁇ 133p53 may be introduced into cells from a patient having a disease of pre-mature aging using a lentiviral vector.
  • Alphavirus genus such as, but not limited to, vectors derived from the Sindbis, Semliki Forest, and Venezuelan Equine Encephalitis viruses, can also be used as viral vectors to deliver a nucleic acid encoding ⁇ 133p53.
  • Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al., J. Virol. (1996) 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No.5,843,723, issued Dec.1, 1998, and Dubensky, Jr., T. W., U.S. Pat. No.5,789,245, issued Aug.4, 1998).
  • the techniques used to obtain ⁇ 133p53 nucleic acids and introduce them into cells may also be used for other nucleic acids as described herein that are employed to extend replicative lifespan of cells from patients with diseases of premature aging.
  • an agent that up-regulates ⁇ 133p53 i.e., that increases the level and/or activity of ⁇ 133p53 in a cell
  • a“small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.
  • the agent may be an agent that targets selective autophagy, the mechanism by which ⁇ 133p53 is degraded in cells of a patient that has a premature aging syndrome, e.g., WS or HGPS, to reduce the selective ⁇ 133p53 autophagy, thereby increasing cellular ⁇ 133p53 levels.
  • a premature aging syndrome e.g., WS or HGPS
  • an agent that up-regulates ⁇ 133p53 in cells from subjects that have a premature aging disease may be an agent that increases the level or activity of a protein that interacts with ⁇ 133p53, such as STUB1 (STIP1 homology and U-Box-containing protein 1, also referred to as CHIP, carboxy terminus of Hsp70- interacting protein).
  • STUB1 has been mapped to 16p33 on human chromosome 16. Protein and nucleic acid sequences for human CHIP are known, as are structural features of the protein that are important to activity.
  • STUB1 has intrinsic E3 ubiquitin ligase activity and promotes ubiquitylation in an in vitro ubiquitylation assay with recombinant proteins ( Jiang et al., J. Biol. Chem.276:42938-42944, 2001). This activity was dependent on the C-terminal U box. Inhibition of endogenous p53 ⁇ expression
  • Inhibitory nucleic acids to p53 ⁇ such as siRNA, shRNA, ribozymes, or antisense molecules, can be synthesized and introduced into cells using methods known in the art. Molecules can be synthesized chemically or enzymatically in vitro (Micura, Agnes Chem. Int. Ed. Emgl.41: 2265–9 (2002); Paddison et al., Proc. Natl. Acad. Sci. USA, 99: 1443–82002) or endogenously expressed inside the cells in the form of shRNAs (Yu et al., Proc. Natl. Acad. Sci.
  • RNA polymerase III U6 or H1, or RNA polymerase II U1 small nuclear RNA promoters
  • Synthetic siRNAs can be delivered by electroporation or by using lipophilic agents (McManus et al., RNA 8, 842– 50 (2002); Kishida et al., J. Gene Med., 6: 105–10 (2004)).
  • plasmid systems can be used to stably express small hairpin RNAs for the suppression of target genes (Dykxhoorn et al., Nat. Rev. Mol. Biol., 4: 457–67 (2003)).
  • Various viral delivery systems have been developed to deliver shRNA-expressing cassettes into cells that are difficult to transfect (Brummelkamp et al., Cancer Cell, 2: 243–7 (2002); Rubinson et al., Nat.
  • siRNAs can also be delivered into live animals.
  • siRNA target sequences should be specific to the gene of interest and have ⁇ 20–50% GC content (Henshel et al., Nucl. Acids Res., 32: 113–20 (2004); G/C at the 5 ⁇ end of the sense strand; A/U at the 5 ⁇ end of the antisense strand; at least 5 A/U residues in the first 7 bases of the 5 ⁇ terminal of the antisense strand; and no runs of more than 9 G/C residues (Ui-Tei et al., Nucl. Acids Res., 3: 936–48 (2004)).
  • RNA polymerase III the polymerase that transcribes from the U6 promoter
  • the preferred target sequence is 5 ⁇ -GN18-3 ⁇ . Runs of 4 or more Ts (or As on the other strand) will serve as terminator sequences for RNA polymerase III and should be avoided. In addition, regions with a run of any single base should be avoided (Czauderna et al., Nucl. Acids Res., 31: 2705–16 (2003)) It has also been generally recommended that the mRNA target site be at least 50–200 bases downstream of the start codon (Sui et al., Proc. Natl. Acad. Sci.
  • Ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy target mRNAs, particularly through the use of hammerhead ribozymes.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.
  • the target mRNA has the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art.
  • Gene targeting ribozymes necessarily contain a hybridizing region complementary to two regions, each of at least 5 and preferably each 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides in length of a target mRNA, e.g., a p53 ⁇ mRNA.
  • ribozymes possess highly specific endoribonuclease activity, which
  • phosphorothioate oligonucleotides can be used. Modifications of the phosphodiester linkage as well as of the heterocycle or the sugar may provide an increase in efficiency. Phophorothioate is used to modify the phosphodiester linkage. An N3'-P5' phosphoramidate linkage has been described as stabilizing oligonucleotides to nucleases and increasing the binding to RNA.
  • PNA linkage is a complete replacement of the ribose and phosphodiester backbone and is stable to nucleases, increases the binding affinity to RNA, and does not allow cleavage by RNAse H. Its basic structure is also amenable to modifications that may allow its optimization as an antisense component. With respect to modifications of the heterocycle, certain heterocycle modifications have proven to augment antisense effects without interfering with RNAse H activity. An example of such modification is C-5 thiazole modification. Finally, modification of the sugar may also be considered.2'-O-propyl and 2'- methoxyethoxy ribose modifications stabilize oligonucleotides to nucleases in cell culture and in vivo. [0077] One of skill understands that inhibitory nucleic acids that target p53 ⁇ can be introduced into cells using principles and methods as described above for the introducing of ⁇ 133p53 nucleic acids into cells.
  • Inhibitory oligonucleotides can be delivered to a cell by direct transfection or transfection and expression via an expression vector.
  • Appropriate expression vectors include mammalian expression vectors and viral vectors, e.g., the illustrative vectors described for delivery of ⁇ 133p53 polynucleotides, into which has been cloned an inhibitory
  • mammalian expression vector can be employed.
  • mammalian expression vectors suitable for siRNA expression are commercially available, for example, from Ambion (e.g., pSilencer vectors), Austin, TX; Promega (e.g., GeneClip, siSTRIKE, SiLentGene), Madison, WI; Invitrogen, Carlsbad, CA; InvivoGen, San Diego, CA; and Imgenex, San Diego, CA.
  • inhibitory RNA sequences are delivered into cells via a viral expression vector.
  • Viral vectors suitable for delivering such molecules to cells include those described above, e.g., adenoviral vectors, adeno-associated vectors, and retroviral vectors (including lentiviral vectors).
  • Agents that down-regulate p53 ⁇ [0079] In some embodiments, an agent that down-regulates p53 ⁇ , i.e., that decreases the level and/or activity of p53 ⁇ in a cell, is administered to a subject that has a disease of premature aging, e.g., WS or HGPS. Such agents are often small organic molecules.
  • a“small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.
  • cells can be treated with an agent that increases SRSF3 expression in cells of patients with disease of premature gaining to inhibit endogenous p53 ⁇ .
  • SRSF3 previously named as SRp20, is the smallest member of the highly conserved serine/arginine-rich splicing factor family and modulates the splicing of numerous genes. It has been shown that down regulation of SRSF3 elevates p53 ⁇ levels (Tang et al., Oncogene 32:2792-2798, 2013).
  • increasing expression of SRSF3 can be achieved by introducing an expression construct that expresses SRSF3, preferably human SRSF3, into cells of patients having a disease of premature aging.
  • an agent that inhibits the function or expression of miR-34a may be used to extend the replicative lifespan of cells from a subject that have a premature aging disease, e.g., WS or HGPS.
  • An exemplary agent useful for this purpose is an antisense oligonucleotide that specifically inactivates the microRNA miR-34a (anti-miR34a).
  • MicroRNAs are endogenous short, e.g., about 21 nucleotides in length, RNA molecules that regulate gene expression post-transcriptionally by interacting with protein-encoding messenger RNAs. miRNAs are similar to short interfering RNAs (siRNAs) in size and function; however, unlike siRNAs, miRNAs are endogenous transcripts encoded by the genome that function to regulate endogenous gene expression. miRNAs possess antisense activity that negatively regulates the expression of genes with sequences that are complementary to specific miRNAs.
  • miR-34a refers to the mature miRNA having the sequence UGGCAGUGUCUUAGCUGGUUGU.
  • nucleic acids that specifically inactivate miR-34a include antisense oligonucleotides.
  • the antisense oligonucleotides may be ribonucleotides or
  • the oligonucleotides may comprise peptide nucleic acids.
  • the oligonucleotide comprises one or more modified nucleotides, such as 2'-O-methyl-sugar modifications. In some embodiments, such inactivating oligonucleotides comprise only modified nucleotides.
  • the oligonculeotides may also contain one or more modifications to the phosphodiester linkage, e.g., may contain one or more phosphorothioate linkages to provide a partial or complete phosphorothioate backbone. Other modifications to enhance stability and improve efficacy may also be employed (see, e.g., U.S. Patent No.6,838,283).
  • the antagonist oligonucleotide may be linked to a cholesterol moiety at its 3’ end for use in vivo.
  • Antagonizing oligonucleotides for inhibiting miR-34a may be about 14 to about 50 nucleotides in length, about 14 to about 30 nucleotides in length, and 14 to about 25 nucleotides in length. In some embodiments, an oligonucleotide may be partially
  • the oligonucleotide need to be 100% complementary to the target nucleotide.
  • the oligonucleotide is at least 75%, 80%, 85%, or 90%, or greater, e.g., 95%, or 100%, complementary to the target sequence, e.g., to the mature miR-34a sequence.
  • an inhibitory nucleic acid used for inhibiting miR-34a may be an inhibitory RNA molecule, such as a ribozyme, siRNAs, or shRNA.
  • the region of the inhibitory RNA that has sequence identity to mature miR-34a typically has 100% identity to the mature sequence, but may also have less than 100% identity, e.g., at least 75%, 80%, 85%, 90%, or 95%, identity to the mature miR-34a sequence. Any number of methods of generating such inhibitory RNA molecules can be used.
  • the oligonucleotides may contain one or more modifications of the phosphodiester linkage as well as of the heterocycle or the sugar to provide an increase in efficiency.
  • inhibitory oligonucleotides can be delivered to a cell by direct transfection or transfection and expression via an expression vector.
  • Appropriate expression vectors include mammalian expression vectors and viral vectors, e.g., the illustrative vectors described for delivery of ⁇ 133p53 polynucleotides, into which has been cloned an inhibitory oligonucleotide with the appropriate regulatory sequences including a promoter to result in expression of the RNA in a host cell.
  • Suitable promoters can be constitutive or development-specific promoters.
  • mammalian expression vector can be employed.
  • mammalian expression vectors suitable for siRNA expression are commercially available, for example, from Ambion (e.g., pSilencer vectors), Austin, TX; Promega (e.g., GeneClip, siSTRIKE, SiLentGene), Madison, WI; Invitrogen, Carlsbad, CA; InvivoGen, San Diego, CA; and Imgenex, San Diego, CA.
  • inhibitory RNA sequences are delivered into cells via a viral expression vector.
  • Viral vectors suitable for delivering such molecules to cells include those described above, e.g., adenoviral vectors, adeno-associated vectors, and retroviral vectors (including lentiviral vectors).
  • Agents can be tested for the ability to up-regulate ⁇ 133p53 or down-regulate p53 ⁇ using any measure of ⁇ 133p53 or p53 ⁇ RNA or protein expression or protein activity.
  • other endpoints may be measured to assess the functional effect of a candidate agent.“Functional effects” include in vitro in vivo, and ex vivo activities. For example, any of a number of methods for the determination and measurement of cell senescence or cell proliferation can be used. In some embodiments, direct measurements of cell proliferation by counting cells may be employed.
  • markers of cellular proliferation e.g., cell markers that are expressed in proliferating cells, such PCNA, or a marker for cellular metabolism such as MTT (see, e.g., Hughes, D., Cell proliferation and apoptosis, Taylor & Francis Ltd, UK (2003)), may also be used to assess cell proliferation and replicative lifespan.
  • PCNA proliferating cells
  • MTT marker for cellular metabolism
  • a number of markers for cell senescence may be used to monitor senescence in the practice of this invention.
  • a common marker is senescence-associated- ⁇ -galactoside (Dimri, G. P. et al., Proc. Natl. Acad. Sci. USA 92:9363 (1995)), although others markers of cell senescence may also be employed.
  • senescence can be measured by direct measurement of telomere length by in situ hybridization or by measurement of age-dependent cellular accumulation of lipofucin in cells (Coates, J. Pathol., 196: 371-3 (2002)).
  • Other markers of senescence include SAHF senescence-associated heterochromatin foci (SAHF) (see, e.g., Methods Mol Biol.965:185–196, 2013).
  • Up-regulation of ⁇ 133p53 is achieved when the measured value relative to the control (untreated with agents to increase ⁇ 133p53 level or activity) is at least 110%, typically at least 120% or at least 150%, or at least 200-at least 500% (i.e., at least two to at least five fold higher relative to the control) in those assays that measure ⁇ 133p53 protein or RNA levels, or that measure an endpoint that is increased when ⁇ 133p53 is increased, e.g., replicative lifespan.
  • up-regulation of ⁇ 133p53 is achieved when the activity value relative to the control is decreased by at least 10% or at least 20%, typically at least 50%, 100%, or 200% or more.
  • an agent that down-regulates p53 ⁇ may also be assessed for functional activity using cell senescence assays, assays for replicative lifespan, or other endpoints as described herein to identify agents that decrease p53 ⁇ .
  • Down-regulation of p53 ⁇ is achieved when the measured value relative to the control (untreated with an agent that decreases the level or activity of p53 ⁇ ) is typically reduced by at least 10%, more often at least 20%, 50%, 75%, 80%, or greater, in those assays that measure p53 ⁇ protein or RNA levels, or that measure an endpoint that is increased when p53 ⁇ is inhibited, e.g., replicative lifespan.
  • inhibition of p53 ⁇ is achieved when the value relative to the control is increased by at least 110%, typically at least 120% or at least 150%, or at least 200-at least 500%.
  • an agent that inhibits miR-34a may also be assessed for functional activity using cell senescence assays, assays for replicative lifespan, or other endpoints as described herein to identify agents that decrease miR-34a.
  • test compound or“drug candidate” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, peptide, circular peptide, lipid, fatty acid, inhibitory RNAs, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly up-regulate ⁇ 133p53.
  • protein oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length)
  • small organic molecule polysaccharide, peptide, circular peptide, lipid, fatty acid, inhibitory RNAs, polynucleotide
  • the test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity.
  • Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • a fusion partner e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • new chemical entities with useful properties are generated by identifying a test compound with some desirable property or activity, e.g., increasing ⁇ 133 levels and/or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • high throughput screening (HTS) methods are employed for such an analysis.
  • assay formats are well-known in the art.
  • stem cells of a patient that has a disease of premature aging may be treated with an agent or otherwise modified to overexpress ⁇ 133p53.
  • the cells are cardiac stem or progenitor cells or endothelial progenitor cells.
  • bone marrow-derived stem/progenitor cells may be treated or otherwise modified to overexpress ⁇ 133p53.
  • stem cells of a patient that has a disease of premature aging may be treated with an agent or otherwise modified to decrease expression of p53 ⁇ .
  • the cells are cardiac stem or progenitor cells or endothelial progenitor cells.
  • bone marrow-derived stem/progenitor cells may be treated or otherwise modified to decrease expression of p53 ⁇ .
  • any suitable method for nucleic acid delivery for transformation of a cell or a tissue of a subject that has a disease of premature aging can be used in the current invention.
  • Such methods include, but are not limited to, direct delivery of nucleic acids, DNA or RNA, such as by ex vivo transfection (Wilson et al., Science, 244:1344-1346, 1989, Nabel and Baltimore, Nature 326:711-713, 1987), optionally with Fugene6 (Roche) or Lipofectamine (Invitrogen), by injection (U.S. Pat.
  • nucleic acids may also be delivered using viral vector systems in which the nucleic acids are delivered in viral particles that infect the desired cells.
  • a composition comprising one or more nucleic acid molecules can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations.
  • a delivery vehicle including liposomes
  • Methods for the delivery of nucleic acid molecules are described, for example, in Gilmore, et al., Curr Drug Delivery (2006) 3:147-5 and Patil, et al., AAPS Journal (2005) 7:E61-E77, each of which are incorporated herein by reference. Delivery of inhibitory RNA molecules is also described in several U.S. Patent Publications, including for example, 2006/0019912; 2006/0014289; 2005/0239687;
  • Nucleic acid molecules can be administered to cells by a variety of methods, including, but not restricted to, encapsulation in liposomes, by iontophoresis, by electroporation, or by incorporation into other vehicles, including biodegradable polymers, hydrogels, cyclodextrins (see, for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO
  • nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
  • polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine PEI-PEG-GAL
  • PEI-PEG-triGAL polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine
  • liposomal transfection reagents of use with this invention include, for example: CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII- tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl- ammoniummethylsulfate) (Boehringer Manheim); Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL); and (5) siPORT (Ambion); HiPerfect (Qiagen);
  • Nucleic acids for administration to a subject are formulated for pharmaceutical administration. While any suitable carrier known may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration.
  • the carrier preferably comprises water, saline, and optionally an alcohol, a fat, a polymer, a wax, one or more stabilizing amino acids or a buffer.
  • General formulation technologies are known to those of skill in the art (see, for example, Remington: The Science and Practice of Pharmacy (20th edition), Gennaro, ed., 2000, Lippincott Williams & Wilkins; Injectable Dispersed Systems: Formulation,
  • Nucleic acid compositions e.g., polynucleotides encoding ⁇ 133p53
  • Multiple administrations can be administered, for example, bi-weekly, weekly, bi-monthly, monthly, or more or less often, as needed, for a time period sufficient to achieve the desired response.
  • the nucleic acid constructs in accordance with the invention are administered to a mammalian host.
  • the mammalian host usually is a human or a primate.
  • the mammalian host can be a domestic animal, for example, canine, feline, lagomorpha, rodentia, rattus, hamster, murine.
  • the mammalian host is an agricultural animal, for example, bovine, ovine, porcine, equine, etc.
  • compositions comprising nucleic acids that encode ⁇ 133p53, or a nucleic acid that inhibits miR-34a, or a nucleic acid that inhibits p53 ⁇ can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art.
  • compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration.
  • the nucleic acids may be administered in an amount sufficient to elicit a therapeutic effect that at least partially arrests or slows one or more symptoms and/or complications of a disease of pre-mature aging, e.g., WS or HGPS.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.
  • the dose of a naked nucleic acid composition is from about 1.0 ng to about 10 mg for a patient.
  • Subcutaneous or intramuscular doses for naked nucleic acid may range from 0.1 ug to 100 ug for a subject.
  • naked DNA or polynucleotide in an aqueous carrier can be injected into tissue, e.g., intramuscularly or intradermally, in amounts of from 10 ⁇ l per site to about 1 ml per site.
  • the concentration of polynucleotide in the formulation is usually from about 0.1 ⁇ g/ml to about 5 mg/ml.
  • the DNA may be administered in ng amounts, for example at a level of 1 to 100 ng.
  • the dose is 0.1 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 1.5 ⁇ g, 2 ⁇ g, 3 ⁇ g, 4 ⁇ g, 5 ⁇ g, 6 ⁇ g, 7 ⁇ g, 8 ⁇ g, 9 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, or 60 ⁇ g of nucleic acids per dose.
  • the dose is in the range of 10 ng to 100 mg, or 50 ng to 100 mg, or 100 ng to 100 mg of nucleic acids per dose. In some specific embodiments, the dose is in the range of 10 pg to 100 mg, or 50 pg to 100 mg, or 100 pg to 100 mg, or 100 pg to 100 ng of nucleic acids per dose.
  • a nucleic acid encoding ⁇ 133p53, or an inhibitory nucleic acid that targets miR-34a, or inhibits p53 ⁇ is delivered using a viral delivery system in which virus particles that comprise the nucleic acid are introduced into the recipient.
  • a nucleic acid encoding ⁇ 133p53 is introduced into cells of a subject having a disease of premature aging, e.g., WS or HGPS, ex vivo.
  • the modified cells may then be cultured, and if desired expanded, and introduced back into the patient.
  • Examples of cells that may be modified ex vivo include stem cells, endothelial progenitor cells (e.g., Yoder Human Endothelial Progenitor Cells, Cold Spring Harb Perspect Med 2012;2:a006692) and cardiac progenitor cells (e.g., U.S. Patent Application Publication No. 20140274765), or bone marrow-derived stem cells and progenitor cells (see, e.g, Lévesque et al, Handb Exp Pharmacol.180:3-36, 2007; Granick et al., Bone Marrow Res. Article ID 165107, 2012).
  • endothelial progenitor cells e.g., Yoder Human Endothelial Progenitor Cells, Cold Spring Harb Perspect Med 2012;2:a006692
  • cardiac progenitor cells e.g., U.S. Patent Application Publication No. 20140274765
  • bone marrow-derived stem cells and progenitor cells see
  • the amount of the therapeutic agent that will be effective in the prevention, treatment and/or management of a disease of premature aging such as WS or HGPS can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend, e.g., on the route of administration, the type of symptoms, and the seriousness of the symptoms, and should be decided according to the judgment of the practitioner and each patient's or subject's circumstances.
  • a nucleic acid that inhibits p53 ⁇ , or a nucleic acid that inhibits miR-34a is introduced into cells of a subject having a disease of premature aging, e.g., WS or HGPS, ex vivo.
  • the modified cells may then be cultured, and if desired expanded, and introduced back into the patient.
  • Examples of cells that may be modified ex vivo include stem cells, endothelial progenitor cells (e.g., Yoder Human Endothelial Progenitor Cells, Cold Spring Harb Perspect Med 2012;2:a006692) and cardiac progenitor cells (e.g., U.S.
  • Patent Application Publication No.20140274765 or bone marrow-derived stem cells and progenitor cells (see, e.g, Lévesque et al, Handb Exp Pharmacol.180:3-36, 2007; Granick et al., Bone Marrow Res. Article ID 165107, 2012).
  • the amount of the therapeutic agent that will be effective in the prevention, treatment and/or management of a disease of premature aging such as WS or HGPS can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend, e.g., on the route of administration, the type of symptoms, and the seriousness of the symptoms, and should be decided according to the judgment of the practitioner and each patient's or subject's circumstances.
  • a zebrafish ortholog of ⁇ 133p53 was recently reported to have gain-of-function roles in transactivating DNA repair genes in response to DNA damage.
  • expression of ⁇ 133p53 might contribute to DNA repair of chronic DNA damage lesions in HGPS cells.
  • the studies described in this example also showed that the DNA repair gene RAD51 is upregulated at the mRNA and protein level in ⁇ 133p53- expressing HGPS cells.
  • Fibroblasts derived from Werner Syndrome (WS) or Hutchinson-Gilford Progeria Syndrome (HGPS) were obtained from Coriell Cell Repository.
  • Cells were grown in complete growth DMEM or MEM (Life Technologies) medium supplemented with 10% fetal bovine serum (Gibco), 2 mM glutamine (Life Technologies) and 50 IU/ml penicillin/streptomycin (Invitrogen). Population doubling levels were calculated as log 10 (number of cells counted after expansion)– log 10 (number of cells seeded)/log 10 2.
  • SA- ⁇ -gal assay Cellular senescence was examined using the Senescence- Associated (SA)- ⁇ -Galactosidase Staining Kit (Cell Signaling) per the manufacturer’s instructions.
  • RNA samples were prepared using a miReasy micro kit (QIAGEN) according to the manufacturer’s instructions.
  • TaqMan Gene Expression Assay (Applied Biosystems) was used with the following set of probe and primers purchased from Applied Biosystems: IL-6 (Hs00174131_m1), p21 (also known as CDKN1A,
  • Hs99999142_m1 miR-34a-5p (cat. no.000426) and RAD51 (Hs00153418_m1).
  • the endogenous control for mRNA expression was B2M ( ⁇ -2-microglobulin, cat. no.4333766) and for microRNA was RNU66 (cat. no.001002). Quantitative data analysis was performed using the ⁇ Ct method.
  • Horseradish peroxidase–conjugated goat anti-mouse (1:5,000) or anti-rabbit (1:5,000) antibodies were used as secondary antibodies. Signals were detected according to standard procedures using ECL detection (Amersham Biosciences) or SuperSignal West Dura Extended Duration system (Pierce Biotechnology). Quantitative analysis of the immunoblot data was performed using ImageJ 1.42q software (see, website at http address rsb.info.nih.gov/ij/).
  • DSBs double strand breaks
  • Cells previously plated onto coverslips were washed with PBS, fixed for 5-10 min on ice-cold methanol at -20°C and subsequently washed with PBS.
  • Cells were permeabilized with 0.25% Triton-X for 5 min on ice, washed with PBS and then blocked in 5% bovine serum albumin (BSA) for 1 h at room temperature. Cells were incubated with primary antibodies overnight at 4 °C.
  • BSA bovine serum albumin
  • the primary antibodies used were: anti- ⁇ H2AX against phosphorylated H2AX (mouse, monoclonal) and anti-RAD51 (rabbit, polyclonal) at a dilution of 1:2000 and 1:1000, respectively.
  • Cells were washed with PBS before incubation for 1h with the the following secondary antibody: Alexa-568-conjugated anti-mouse and Alexa-488-conjugated anti-rabbit at a dilution of 1:400 (Life Technologies).4’,6-diamidino-2-phenylindole was used to stain the nucleus. Coverslips were mounted on to slides with FluorSave mounting medium (Chemicon).
  • a Zeiss LSM 780 confocal microscope was used to take images. Quantification of ⁇ H2AX-positive DSBs was performed using the automated software ImagePremier Pro to detect and count nuclear ⁇ H2AX-positive foci. Co-localization of ⁇ H2AX-positive DSBs with RAD51 was quantified and DSBs were scored as RAD51-positive (RAD51+ve) or RAD51-negative (RAD51-ve). At least 100 cells were counted per experiment.

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Abstract

La présente invention concerne des thérapies qui modulent les isoformes p53 existant à l'état naturel pour le traitement de maladies du vieillissement prématuré.
PCT/US2016/034821 2015-05-29 2016-05-27 Extension de durée de vie réplicative dans des maladies de vieillissement prématuré à l'aide d'isoformes p53 WO2016196366A1 (fr)

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US20190365822A1 (en) * 2017-02-01 2019-12-05 Aal Scientifics, Inc. CARDIAC PROGENITOR CELLS HAVING ENHANCED p53 EXPRESSION AND USES THEREOF

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