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WO2018130518A1 - Procédés et composition pharmaceutique d'induction de la sénescence dans des cellules cancéreuses - Google Patents

Procédés et composition pharmaceutique d'induction de la sénescence dans des cellules cancéreuses Download PDF

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Publication number
WO2018130518A1
WO2018130518A1 PCT/EP2018/050446 EP2018050446W WO2018130518A1 WO 2018130518 A1 WO2018130518 A1 WO 2018130518A1 EP 2018050446 W EP2018050446 W EP 2018050446W WO 2018130518 A1 WO2018130518 A1 WO 2018130518A1
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Prior art keywords
malignant
carcinoma
cell
adenocarcinoma
tumor
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PCT/EP2018/050446
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English (en)
Inventor
Eric Chevet
Delphine FESSART
Frédéric DELOM
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université De Rennes 1
Université De Bordeaux
Institut Bergonié
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Publication of WO2018130518A1 publication Critical patent/WO2018130518A1/fr

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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • 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
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/04—Intramolecular oxidoreductases (5.3) transposing S-S bonds (5.3.4)
    • C12Y503/04001—Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme

Definitions

  • the present invention relates to methods and pharmaceutical composition for inducing senescence in cancer cells.
  • AGR2 anterior Gradient Protein 2
  • AGR2 is an endoplasmic reticulum protein involved in oncogenesis
  • AGR2 was overexpressed in different cancers (Chevet et al. 2013) like prostate, lung, breast, ...
  • AGR2 is involved in many biological processes such as cell proliferation, adhesion, anti-apoptosis, senescence and cell cycle regulation (Chevet et al. 2013).
  • siRNA siRNA
  • the present invention relates to methods and pharmaceutical composition for inducing senescence in cancer cells.
  • the present invention is defined by the claims.
  • the inventors demonstrate that knockdown of AGR2 by CRISPRCas9 system leads to a senescence process in cancer cells and thus represent a promising tool for the treatment of cancer.
  • the first object of the present invention relates to a method of inducing senescence in a population of cancer cells of a subject comprising contacting the population of cancer cells with a gene editing complex comprising a CRISPR-associated endonuclease and a guide RNA, wherein the guide RNA is complementary to a target nucleic acid sequence within the gene encoding for AGR2.
  • the population of cancer cells is a population of bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus cancer cells.
  • Senescence has its general mean in the art and refers to the permanent cessation of DNA replication and cell growth that is not reversible by growth factors. Senescence can be characterized by certain morphological features including, but not limited to, increased size, flattened morphology, increased granularity, and senescence-associated ⁇ - galactosidase activity (SA- ⁇ -gal).
  • AGR2 has its general meaning in the art and refers to the gene encoding for the anterior gradient 2, protein disulphide isomerase family member (Gene ID: 10551).
  • the genomic sequence is referenced inthe NCBI database under the NC 000007.14 accession number.
  • An exemplary nucleic sequence is represented by the NCBI reference sequence NM 006408.3 and an exemplary amino acid sequence is represented by the NCBI reference sequence NP 006399.1.
  • the population of cancer cells is bringing into contact with an isolated nucleic acid sequence encoding a CRISPR-associated endonuclease and an isolated nucleic acid sequence encoding one or more guide RNA.
  • an isolated nucleic acid sequence includes nucleic acids encoding a CRISPR-associated endonuclease, e.g., Cas9, and one or more guide RNA that is complementary to a target nucleic acid sequence within the AGR2 gene.
  • An "isolated" nucleic acid can be, for example, a naturally-occurring DNA molecule or a fragment thereof, provided that at least one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restriction endonuclease treatment).
  • An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote.
  • an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid.
  • Isolated nucleic acid molecules can be produced by standard techniques.
  • CRISPR-associated endonuclease has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
  • the CRISPR/Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids).
  • Three types (I-III) of CRISPR systems have been identified.
  • CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements.
  • CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA).
  • the CRISPR-associated endonuclease belongs to the type II CRISPR/Cas system and has strong endonuclease activity to cut target DNA.
  • Cas9 is guided by a mature crRNA that contains about 20 base pairs (bp) of unique target sequence (called spacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease Ill-aided processing of pre-crRNA.
  • spacer unique target sequence
  • tracrRNA trans-activated small RNA
  • the crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA.
  • Cas9 recognizes a trinucleotide (NGG) protospacer adjacent motif (PAM) to specify the cut site (the 3rd nucleotide from PAM).
  • the crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion small guide RNA (sgRNA) via a synthetic stem loop to mimic the natural crRNA/tracrRNA duplex.
  • sgRNA like shRNA, can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6 or HI -promoted RNA expression vector, although cleavage efficiencies of the artificial sgRNA are lower than those for systems with the crRNA and tracrRNA expressed separately.
  • the CRISPR-associated endonuclease can be a Cas9 nuclease.
  • the Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyrogenes sequence.
  • the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as thermophilus; Pseudomona aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microogranisms.
  • the wild type Streptococcus pyrogenes Cas9 sequence can be modified.
  • the nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, i.e., "humanized.”
  • a humanized Cas9 nuclease sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765.
  • the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, MA).
  • the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GL669193757; KM099232.1; GL669193761; or KM099233.1 GL669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, MA).
  • the Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations).
  • One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution).
  • a biologically active variant of a Cas9 polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a wild type Cas9 polypeptide.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
  • the Cas9 nuclease sequence can be a mutated sequence.
  • the Cas9 nuclease can be mutated in the conserved FiNH and RuvC domains, which are involved in strand specific cleavage.
  • an aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single- stranded breaks, and the subsequent preferential repair through HDR can potentially decrease the frequency of unwanted indel mutations from off- target double-stranded breaks.
  • polypeptides that are biologically active variants of a CRISPR-associated endonuclease can be characterized in terms of the extent to which their sequence is similar to or identical to the corresponding wild-type polypeptide.
  • sequence of a biologically active variant can be at least or about 80% identical to corresponding residues in the wild-type polypeptide.
  • a biologically active variant of a CRISPR- associated endonuclease can have an amino acid sequence with at least or about 80% sequence identity (e.g., at least or about 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a CRISPR- associated endonuclease or to a homolog or ortholog thereof.
  • a biologically active variant of a CRISPR-associated endonuclease polypeptide will retain sufficient biological activity to be useful in the present methods.
  • the biologically active variants will retain sufficient activity to function in targeted DNA cleavage.
  • the biological activity can be assessed in ways known to one of ordinary skill in the art and includes, without limitation, in vitro cleavage assays or functional assays.
  • the term "one or more guide RNA” refers to the RNAs that guide the insertion or deletion of residues.
  • the guide RNA is used for recruiting Cas9 to specific genomic loci.
  • the guide RNA can be a sequence complementary to a coding or a non-coding sequence.
  • the guide RNA can be sequence complimentary to an AGR2 sequence, such as a protein coding sequence or a regulatory sequence.
  • the complementary target sequence to the guide RNA sequence includes a proto-spacer adjacent motif (PAM) which is sensed by the Cas9.
  • the sequence of the PAM can vary depending upon the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system derived from S.
  • the target DNA typically immediately precedes a 5'-NGG proto-spacer adjacent motif (PAM).
  • PAM 5'-NGG proto-spacer adjacent motif
  • the PAM sequence can be AGG, TGG, CGG or GGG.
  • Other Cas9 orthologs may have different PAM specificities.
  • the specific sequence of the guide RNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency and complete ablation of the AGR2 genome.
  • the length of the guide RNA sequence can vary from about 19 to about 60 or more nucleotides, for example about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 or more nucleotides.
  • Useful selection methods identify regions having extremely low homology between the foreign viral genome and host cellular genome including endogenous retroviral DNA, include bioinformatic screening using 12-bp+NGG target-selection criteria to exclude off-target human transcriptome or (even rarely) untranslated-genomic sites, Sanger sequencing and SURVEYOR assay, to identify and exclude potential off-target effects.
  • the guide RNA sequence can be configured as a single sequence or as a combination of one or more different sequences, e.g., a multiplex configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs.
  • Exemplary guide RNA sequences include SEQ ID NO: 1 and SEQ ID NO:2.
  • the RNA molecules e.g. crRNA, tracrRNA, gRNA are engineered to comprise one or more modified nucleobases.
  • modified nucleobases known modifications of RNA molecules can be found, for example, in Genes VI, Chapter 9 ("Interpreting the Genetic Code"), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne, eds. (1998, ASM Press, Washington DC).
  • Modified RNA components include the following: 2'-0-methylcytidine; 5-methylcytidine; 5,2'-0- dimethylcytidine; 5-hydroxymethylcytidine; 5-formylcytidine; 3-methylcytidine; 2- thiocytidine; lysidine; 2'-0-methyluridine; 2-thiouridine; 2-thio-2'-0-methyluridine; 3,2'-0- dimethyluridine; 4-thiouridine; 5-methyl-2-thiouridine; 5-hydroxyuridine; 5-methoxyuridine; dihydrouridine; inosine; 2'0-methyl inosine; 1 -methyl inosine; and 1 -methyl guanosine.
  • the CRISPR-associated endonuclease and the guide RNA are provided to the population of cancer cells through expression from one or more expression vectors coding therefore.
  • the CRISPR endonuclease can be encoded by the same nucleic acid as the guide RNA sequences.
  • the CRISPR endonuclease can be encoded in a physically separate nucleic acid from the guide RNA sequences or in a separate vector.
  • vector is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs.
  • the term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors.
  • An "expression vector” is a vector that includes a regulatory region. A wide variety of host/expression vector combinations may be used to express the nucleic acid sequences described herein.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculo viruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).
  • the vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers.
  • a marker gene can confer a selectable phenotype on a host cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin).
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FlagTM tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • hemagglutinin or FlagTM tag (Kodak, New Haven, CT) sequences
  • FlagTM tag Kodak, New Haven, CT sequences
  • Additional expression vectors also can include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified
  • Yeast expression systems can also be used.
  • the non-fusion pYES2 vector (Xbal, Sphl, Shol, Notl, GstXI, EcoRI, BstXI, BamHl, Sad, Kpnl, and Hindlll cloning sites; Invitrogen) or the fusion pYESHisA, B, C (Xbal, Sphl, Shol, Notl, BstXI, EcoRI, BamHl, Sad, Kpnl, and Hindlll cloning sites, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the invention.
  • a yeast two-hybrid expression system can also be prepared in accordance with the invention.
  • the vector can also include a regulatory region.
  • regulatory region refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.
  • operably linked refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence.
  • the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site.
  • a promoter typically comprises at least a core (basal) promoter.
  • a promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • control element such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • the choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.
  • Vectors include, for example, viral vectors (such as adenoviruses (“Ad”), adeno- associated viruses (AAV), and vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macro molecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • a “recombinant viral vector” refers to a viral vector comprising one or more heterologous gene products or sequences. Since many viral vectors exhibit size-constraints associated with packaging, the heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-defective, requiring the deleted function(s) to be provided in trans during viral replication and encapsidation (by using, e.g., a helper virus or a packaging cell line carrying gene products necessary for replication and/or encapsidation).
  • Suitable nucleic acid delivery systems include recombinant viral vector, typically sequence from at least one of an adenovirus, adenovirus-associated virus (AAV), helper- dependent adenovirus, retrovirus, or hemagglutinating virus of Japan- liposome (HVJ) complex.
  • the vector is an AAV vector.
  • AAV vector means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences.
  • Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines.
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components.
  • a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media.
  • the media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer.
  • Retroviral vectors are able to infect a broad variety of cell types.
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection.
  • Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV).
  • Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S.
  • the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell.
  • the gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
  • Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
  • This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell.
  • Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest.
  • the env preferably is an amphotropic envelope protein that allows transduction of cells of human and other species.
  • the nucleic acid molecule or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • nucleic acid sequence is a "promoter” sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
  • Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters”.
  • the method of the present invention is particularly suitable for the treatment of cancer.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors
  • cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • cancer further encompasses both primary and metastatic cancers.
  • cancers that may treated by methods and compositions of the invention include, but are not limited to neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lympho epithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary a
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • a “therapeutically effective amount” is meant a sufficient amount of the isolated nucleic acid (or the vector containing the isolated nucleic acid) to treat the disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 100 mg/kg of body weight per day.
  • compositions of the invention can be prepared in a variety of ways known to one of ordinary skill in the art. Regardless of their original source or the manner in which they are obtained, the compositions of the invention can be formulated in accordance with their use.
  • the nucleic acids and vectors described above can be formulated within compositions for application to cells in tissue culture or for administration to a patient or subject.
  • Any of the pharmaceutical compositions of the invention can be formulated for use in the preparation of a medicament, and particular uses are indicated below in the context of treatment, e.g., the.
  • any of the nucleic acids and vectors can be administered in the form of pharmaceutical compositions.
  • compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal, intravesical and rectal delivery), pulmonary ⁇ e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular intravesical injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration.
  • Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump.
  • Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • This invention also includes pharmaceutical compositions which contain, as the active ingredient, nucleic acids and vectors described herein in combination with one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.
  • the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container.
  • an excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient.
  • the type of diluent can vary depending upon the intended route of administration.
  • the nucleic acid sequences of the invention can be delivered to an appropriate cell of a subject.
  • a polymeric, biodegradable microparticle or microcapsule delivery vehicle sized to optimize phagocytosis by phagocytic cells such as macrophages.
  • a polymeric, biodegradable microparticle or microcapsule delivery vehicle sized to optimize phagocytosis by phagocytic cells such as macrophages.
  • PLGA poly-lacto-co-glycolide
  • microparticles approximately 1-10 ⁇ in diameter can be used.
  • the polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide. Once released, the DNA is expressed within the cell.
  • a second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro-particle through biodegradation.
  • These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5 ⁇ and preferably larger than 20 ⁇ ).
  • Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods.
  • the nucleic acids can be incorporated alone into these delivery vehicles or co -incorporated with tissue-specific antibodies.
  • Poly-L-lysine binds to a ligand that can bind to a receptor on target cells. Delivery of "naked DNA" (i.e., without a delivery vehicle) to an intramuscular, intradermal,, intravesical or subcutaneous site, is another means to achieve in vivo expression.
  • the nucleic acid sequence encoding an isolated nucleic acid sequence comprising a sequence encoding a CRISPR-associated endonuclease and a guide RNA is operatively linked to a promoter or enhancer-promoter combination.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 CRISPR/Cas9-mediated knockout of AGR2 in H1838 cells induce senescence.
  • A Schematic representation of the designed guide RNAs (gRNA) targeting exon 1 of the AGR2 gene.
  • B Representative Western blot for AGR2, Calnexin (CANX) and GAPDH protein expression in control cells (HI 838) and after CRISPR/Cas9-mediated AGR2 knockout.
  • C Analysis by immunofluorescence of SA- gal activity in in control cells (HI 838) and after CRISPR/Cas9-mediated AGR2 knockout. Scale bars, 100 ⁇ .
  • AGR2 is essential for cancer tumorigenesis.
  • HBEC-EV empty vector
  • AGR2 containing vector HBEC-AGR2
  • AGR2 co-localized with calnexin (CANX), an ER resident type I integral membrane protein and BiP (GRP78), a soluble HSP70 molecular chaperone located in the lumen of the ER.
  • CANX calnexin
  • GPP78 an ER resident type I integral membrane protein and BiP
  • AGR2 did not co-localize with GM130, a cis- Golgi marker.
  • AGR2 Increased AGR2 expression in these cells (HBEC-AGR2) was also confirmed using Western blotting.
  • AGR2 overexpression enhanced cell proliferation and increased the formation of organoids, thereby indicating that the expression of AGR2 dictates organoid- initiating frequency.
  • Inhibition of AGR2 by CRISPR-Cas9 system promotes Senescence in Cancer Cells
  • the use of AGR2- mediated CRISPR knock-down for engagement of senescence may represent a key target for therapeutic intervention in the eradication of cancer. Therefore, the present invention relates the use AGR2-CRISPR mediated knock-down to deplete Anterior- gradient 2 (AGR2) and inducing senescence for treating cancer.
  • AGR2 Anterior- gradient 2

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Abstract

La présente invention concerne des procédés et une composition pharmaceutique d'induction de la sénescence dans les cellules cancéreuses. Les inventeurs mettent en évidence que l'inactivation d'AGR2 (gradient antérieur 2) par le système CRISPRCas9 conduit à un processus de sénescence dans les cellules cancéreuses et représente ainsi un outil prometteur pour le traitement du cancer. En particulier, la présente invention concerne un procédé d'induction de la sénescence dans une population de cellules cancéreuses d'un sujet comprenant la mise en contact de la population de cellules cancéreuses avec un complexe d'édition génique comprenant une endonucléase associée à CRISPR et un ARN guide, l'ARN guide étant complémentaire d'une séquence d'acide nucléique cible dans le gène codant pour AGR2.
PCT/EP2018/050446 2017-01-10 2018-01-09 Procédés et composition pharmaceutique d'induction de la sénescence dans des cellules cancéreuses WO2018130518A1 (fr)

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