[go: up one dir, main page]

WO2024130151A1 - Compositions et méthodes pour traiter une grande chimiorésistance par l'intermédiaire de composants régulateurs spécifiques de la chimiorésistance - Google Patents

Compositions et méthodes pour traiter une grande chimiorésistance par l'intermédiaire de composants régulateurs spécifiques de la chimiorésistance Download PDF

Info

Publication number
WO2024130151A1
WO2024130151A1 PCT/US2023/084338 US2023084338W WO2024130151A1 WO 2024130151 A1 WO2024130151 A1 WO 2024130151A1 US 2023084338 W US2023084338 W US 2023084338W WO 2024130151 A1 WO2024130151 A1 WO 2024130151A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
substituting positions
reverse complement
substituting
positions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2023/084338
Other languages
English (en)
Inventor
Eric B. Kmiec
Byung-Chun Yoo
Kelly H. BANAS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Christiana Care Gene Editing Institute Inc
Original Assignee
Christiana Care Gene Editing Institute Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Christiana Care Gene Editing Institute Inc filed Critical Christiana Care Gene Editing Institute Inc
Publication of WO2024130151A1 publication Critical patent/WO2024130151A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/1135—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 oncogenes or tumor suppressor genes
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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

Definitions

  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated genes
  • MDR Multidrug resistance
  • P-gp Permeability glycoprotein 1
  • MDR1 multidrug resistance protein 1
  • ABSB1 ATP-binding cassette sub-family B member 1
  • Lung carcinomas are the leading cause of cancer deaths in the United States and worldwide in both men and women. Chemotherapy for non-small-cell lung carcinoma (NSCLC), which accounts for approximately 85% of lung cancer cases, remains marginally effective. The major contributing factor to the failure of chemotherapy in lung cancer is the development of drug resistance.
  • Cisplatin (CDDP) is the current and most widely used chemotherapeutic agent in several solid malignancies such as lung and ovarian cancers. It is a platinum compound that binds to and crosslinks DNA, thus inducing apoptosis in cancer cells.
  • ROS Reactive Oxygen Species
  • the complex cellular response against ROS is mainly mediated through the antioxidant 2 ME147020341v.2 130949-01420 response element (ARE), the nuclear factor erythroid-related factor-2 (Nrf2) and the small-Maf-proteins family.
  • ARE antioxidant 2 ME147020341v.2 130949-01420 response element
  • Nrf2 nuclear factor erythroid-related factor-2
  • small-Maf-proteins family an overexpression of these elements could be responsible of the acquired resistance to cisplatin.
  • Cancer chemotherapy aims to treat cancer with chemicals that maximize the killing of neoplastic cells while minimizing the killing of most/all other host cells (Chabner et al., Nat.
  • chemotherapeutic agents used today include dexamethasone, cisplatin, etoposide, cytosine arabinoside, taxol, 5- fluorouracil, doxorubicin, topotecan, and bleomycin. Most chemotherapeutic agents induce programmed cell death/apoptosis via the endogenous or intrinsic mitochondrial pathway. Chemotherapy is able to induce apoptosis in four stages (Bold et al., Surg.
  • CRISPR/Cas has great potential as a tool for use in gene therapy.
  • One of the key advantages of the technology is its ability to specifically bind to a given genomic sequence with high precision and induce double-stranded breaks, resulting in frame- shifting indels that can lead to a knockout.
  • One aspect is for a DNA sequence comprising a first polynucleotide encoding a Cas endonuclease of a CRISPR/Cas system, the first polynucleotide operably linked to 3 ME147020341v.2 130949-01420 a regulatory region that is activated by a second polynucleotide targeted by the CRISPR/Cas system, wherein expression of the CRISPR/Cas system reduces expression of the second polynucleotide.
  • the DNA sequence further comprises a third polynucleotide encoding one or more guide RNAs (gRNAs) of the CRISPR/Cas system, the one or more gRNAs having a binding site on the second polynucleotide; in some embodiments, each gRNA is operably linked to one or more promoters; in some embodiments, the one or more gRNAs each comprise a trans- activated small RNA (tracrRNA) and a CRISPR RNA (crRNA); and in some embodiments, the one or more gRNAs each are a single guide RNA (sgRNA).
  • gRNAs guide RNAs
  • the regulatory region comprises one or more promoters; and in some embodiments, the one or more promoters are RNA polymerase I, RNA polymerase II, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase (PGK) promoter, a minimal promoter, or a combination thereof.
  • RSV Rous sarcoma virus
  • PGK phosphoglycerol kinase
  • the regulatory region comprises one or more response elements; and in some embodiments, the one or more response elements are an antioxidant response element, a cAMP response element, a B recognition element, an AhR-responsive element, a dioxin-responsive element, a xenobiotic-responsive element, an estrogen response element, an androgen response element, a serum response element, a retinoic acid response element, a peroxisome proliferator hormone response element, a metal-responsive element, a DNA damage response element, an IFN-stimulated response element, an ROR-response element, a glucocorticoid response element, a calcium-response element CaRE1, an antioxidant response element, a p53 response element, a thyroid hormone response element, a growth hormone response element, a sterol response element, a polycomb response element, a Vitamin D response element, a Rev response element, or a combination thereof.
  • the one or more response elements are an antioxidant response element, a cAMP response element,
  • the Cas endonuclease is a class 2 Cas endonuclease; and in some embodiments, the Cas endonuclease is Cas9, Cas12a, Cas12b1, Cas12b2, Cas12c, Cas12d, Cas12e, Cas12f1, Cas12f2, Cas12f3, Cas12g, Cas12h, 4 ME147020341v.2 130949-01420 Cas12i, Cas12k, Cas12j, Cas13a, Cas13b1, Cas13b2, Cas13c, Cas13d, c2c4, c2c8, c2c9, or c2c10.
  • the second polynucleotide encodes NRF2, EGFR, or KRAS; in some embodiments, NRF2 is a variant NRF2; and in some embodiments, the variant NRF2 comprises: (a) one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:2 substituting positions 205-227 of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:5 substituting positions 230-252 of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:
  • reduced expression of the second polynucleotide results in reduced regulatory region activity and reduced expression of the first polynucleotide.
  • Another aspect is for a vector comprising the aforementioned DNA sequence.
  • a further aspect is for a host cell comprising the aforementioned DNA sequence or the aforementioned vector.
  • Another aspect is for a pharmaceutical composition comprising the aformentioned DNA sequence, the aforementioned vector, or the aforementioned host cell.
  • An additional aspect is for a method of regulating Cas endonuclease expression comprising introducing into a cell a vector comprising a first polynucleotide encoding a Cas endonuclease of a CRISPR/Cas system, the first polynucleotide operably linked to a regulatory region that is activated by a second polynucleotide targeted by the CRISPR/Cas system, whereby expression of the CRISPR/Cas system in the cell reduces expression of the second polynucleotide.
  • the vector further comprises a third polynucleotide encoding one or more gRNAs of the CRISPR/Cas system, the one or more gRNAs having a binding site on the second polynucleotide; and in some embodiments, each gRNA is operably linked to one or more promoters.
  • the method further comprises introducing into a cell a second vector comprising a third polynucleotide encoding one or more gRNAs of the CRISPR/Cas system, the one or more gRNAs having a binding site on the second polynucleotide; and in some embodiments, each gRNA is operably linked to one or more promoters.
  • the one or more gRNAs each comprise a tracrRNA and a crRNA; and in some embodiments, the one or more gRNAs each are an sgRNA.
  • the regulatory region comprises one or more promoters; and in some embodiments, the one or more promoters are RNA polymerase I, RNA polymerase II, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, SV40 8 ME147020341v.2 130949-01420 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase (PGK) promoter, a minimal promoter, or a combination thereof.
  • RSV Rous sarcoma virus
  • PGK phosphoglycerol kinase
  • the regulatory region comprises one or more response elements; and in some embodiments, the one or more response elements are an antioxidant response element, a cAMP response element, a B recognition element, an AhR-responsive element, a dioxin-responsive element, a xenobiotic-responsive element, an estrogen response element, an androgen response element, a serum response element, a retinoic acid response element, a peroxisome proliferator hormone response element, a metal-responsive element, a DNA damage response element, an IFN-stimulated response element, an ROR-response element, a glucocorticoid response element, a calcium-response element CaRE1, an antioxidant response element, a p53 response element, a thyroid hormone response element, a growth hormone response element, a sterol response element, a polycomb response element, a Vitamin D response element, a Rev response element, or a combination thereof.
  • the one or more response elements are an antioxidant response element, a cAMP response element,
  • the Cas endonuclease is a class 2 Cas endonuclease; and in some embodiments, the Cas endonuclease is Cas9, Cas12a, Cas12b1, Cas12b2, Cas12c, Cas12d, Cas12e, Cas12f1, Cas12f2, Cas12f3, Cas12g, Cas12h, Cas12i, Cas12k, Cas12j, Cas13a, Cas13b1, Cas13b2, Cas13c, Cas13d, c2c4, c2c8, c2c9, or c2c10.
  • the second polynucleotide encodes NRF2, EGFR, or KRAS; in some embodiments, NRF2 is a variant NRF2; and in some embodiments, the variant NRF2 comprises: (a) one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:2 substituting positions 205-227 of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:5 substituting positions 230-252 of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ 9 ME147020341v.
  • reduced expression of the second polynucleotide results in reduced regulatory region activity and reduced expression of the first polynucleotide.
  • Another aspect is for amethod of treating cancer comprising introducing into a cancer cell a vector comprising a first polynucleotide encoding a Cas endonuclease of a CRISPR/Cas system, the first polynucleotide operably linked to a regulatory region that is activated by a second polynucleotide targeted by the CRISPR/Cas system, whereby expression of the CRISPR/Cas system in the cell reduces expression of the second polynucleotide.
  • the vector further comprises a third polynucleotide encoding one or more gRNAs of the CRISPR/Cas system, the one or 12 ME147020341v.2 130949-01420 more gRNAs having a binding site on the second polynucleotide; and in some embodiments, each gRNA is operably linked to one or more promoters.
  • the method further comprises introducing into a cell a second vector comprising a third polynucleotide encoding one or more gRNAs of the CRISPR/Cas system, the one or more gRNAs having a binding site on the second polynucleotide; and in some embodiments, each gRNA is operably linked to one or more promoters.
  • the one or more gRNAs each comprise a tracrRNA and a crRNA; and in some embodiments, the one or more gRNAs each are an sgRNA.
  • the regulatory region comprises one or more promoters; and in some embodiments, the one or more promoters are RNA polymerase I, RNA polymerase II, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase (PGK) promoter, a minimal promoter, or a combination thereof.
  • RSV Rous sarcoma virus
  • PGK phosphoglycerol kinase
  • the regulatory region comprises one or more response elements; and in some embodiments, the response element is an antioxidant response element, a cAMP response element, a B recognition element, an AhR-responsive element, a dioxin-responsive element, a xenobiotic-responsive element, an estrogen response element, an androgen response element, a serum response element, a retinoic acid response element, a peroxisome proliferator hormone response element, a metal-responsive element, a DNA damage response element, an IFN-stimulated response element, an ROR-response element, a glucocorticoid response element, a calcium-response element CaRE1, an antioxidant response element, a p53 response element, a thyroid hormone response element, a growth hormone response element, a sterol response element, a polycomb response element, a Vitamin D response element, a Rev response element, or a combination thereof.
  • the response element is an antioxidant response element, a cAMP response element, a B recognition element,
  • the Cas endonuclease is a class 2 Cas endonuclease; and in some embodiments, the Cas endonuclease is Cas9, Cas12a, Cas12b1, Cas12b2, Cas12c, Cas12d, Cas12e, Cas12f1, Cas12f2, Cas12f3, Cas12g, Cas12h, 13 ME147020341v.2 130949-01420 Cas12i, Cas12k, Cas12j, Cas13a, Cas13b1, Cas13b2, Cas13c, Cas13d, c2c4, c2c8, c2c9, or c2c10.
  • the second polynucleotide encodes NRF2, EGFR, or KRAS; in some embodiments, NRF2 is a variant NRF2; and in some embodiments, the variant NRF2 comprises: (a) one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:2 substituting positions 205-227 of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:5 substituting positions 230-252 of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:
  • the cancer is a chemoresistant tumor.
  • reduced expression of the second polynucleotide results in reduced regulatory region activity and reduced expression of the first polynucleotide.
  • a further aspect is for a composition for use in treating cancer comprising a DNA sequence comprising a first polynucleotide encoding a Cas endonuclease of a CRISPR/Cas system, the first polynucleotide operably linked to a regulatory region that is activated by a second polynucleotide targeted by the CRISPR/Cas system, wherein expression of the CRISPR/Cas system reduces expression of the second polynucleotide.
  • the composition further comprises a third polynucleotide encoding one or more gRNAs of the CRISPR/Cas system, the one or more gRNAs having a binding site on the second polynucleotide; in some embodiments, each gRNA is operably linked to one or more promoters; in some embodiments, the one or more gRNAs each comprise a tracrRNA and a crRNA; and in some embodiments, the one or more gRNAs each are an sgRNA.
  • the regulatory region comprises one or more promoters; and in some embodiments, the one or more promoters are RNA polymerase I, RNA polymerase II, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase (PGK) promoter, a minimal promoter, or a combination thereof.
  • RSV Rous sarcoma virus
  • SV40 promoter SV40 promoter
  • dihydrofolate reductase promoter phosphoglycerol kinase (PGK) promoter
  • PGK phosphoglycerol kinase
  • the regulatory region comprises one or more response elements; and in some embodiments, the one or more response elements are an antioxidant response element, a cAMP response element, a B recognition element, an AhR-responsive element, a dioxin-responsive element, a xenobiotic-responsive element, an estrogen response element, an androgen response element, a serum response element, a retinoic acid response element, a peroxisome proliferator hormone response element, a metal-responsive element, a DNA damage response element, an IFN-stimulated response element, an ROR-response element, a glucocorticoid response element, a calcium-response element CaRE1, an antioxidant 17 ME147020341v.2 130949-01420 response element, a p53 response element, a thyroid hormone response element, a growth hormone response element, a sterol response element, a polycomb response element, a Vitamin D response element, a Rev response element, or a combination thereof.
  • the one or more response elements
  • the Cas endonuclease is a class 2 Cas endonuclease; and in some embodiments, the Cas endonuclease is Cas9, Cas12a, Cas12b1, Cas12b2, Cas12c, Cas12d, Cas12e, Cas12f1, Cas12f2, Cas12f3, Cas12g, Cas12h, Cas12i, Cas12k, Cas12j, Cas13a, Cas13b1, Cas13b2, Cas13c, Cas13d, c2c4, c2c8, c2c9, or c2c10.
  • the second polynucleotide encodes NRF2, EGFR, or KRAS; in some embodiments, NRF2 is a variant NRF2; and in some embodiments, the variant NRF2 comprises: (a) one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:2 substituting positions 205-227 of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:5 substituting positions 230-252 of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:
  • the cancer is a chemoresistant tumor.
  • reduced expression of the second polynucleotide results in regulatory region element activity and reduced expression of the first polynucleotide.
  • An additional aspect is for a vector comprising the aforementioned composition.
  • Another aspect is for a host cell comprising the aforementioned composition or the aformentioned vector.
  • Each domain carries a specific function of the protein overall. Transcriptional activity is regulated by domains Neh4, Neh5 and Neh1.
  • the Neh1 domain is responsible for binding to an ARE within a downstream target gene.
  • the Neh4 and Neh5 are responsible for recruiting RNA polymerase II through various protein complexes to initiate transcription of the downstream target gene. (Modified from Guerrero-Hue et al.2020).
  • the top panel (A) depicts a NRF2- regulated promoter activating transcription of downstream target genes (ie. NQO1, GSTA, HMOX1).
  • the bottom panel (B) depicts a NRF2-regulated promoter activating transcription of the vector to express CRISPR/Cas nuclease.
  • FIG. 1 Schematic diagram of CompleteKill. NQO1 promoter region was cloned into an adenoviral plasmid to replace a chicken beta actin (CAG) promoter driving eSpCas9 expression.
  • the adenoviral plasmid contains three promoters, U6, H1 and 7SK, driving NRF2-targeting gRNA expression.
  • Figure 4. Genomic Structure of NQO1. The figure depicts the 5' UTR and promoter region of NQO1. The ARE sequence is shown in light blue with respective primers used to amplify the promoter region.
  • Figure 5. Vector map of pGL3-NQO1pro.
  • the NQO1 promoter region was ligated into a promoterless luciferase expression plasmid.
  • the NQO1 promoter region is shown in white with the respective elements of the luciferase expression plasmid.
  • Figure 6. Vector map of pGL3-CAGpro.
  • the CAG promoter was ligated into a promoterless luciferase expression plasmid.
  • the CAG promoter is shown in grey with the respective elements of the luciferase expression plasmid.
  • Figure 7. Luciferase Activity after transfection of pGL3-control, pGL3- CAGpro and pGL3-NQO1pro vectors.
  • the graph displays raw luminescence values of each respective plasmid transfected by nucleofection or lipofection. Luciferase activity was determined 48 and 72 hours post transfection and displayed as a raw luminescent value.
  • Figure 8. Vector map of pAd-U6H17SK-R34G-nopro-Cas9.
  • the plasmid contains NRF2-targeting gRNAs driven by U6, H1 and 7SK promoters followed by eSpCas9 open reading frame without a promoter.
  • Figure 9 Vector map of pAd-U6H17SK-R34G-NQO1pro-Cas9.
  • the NQO1 promoter region was ligated into an adenoviral plasmid preceding the Cas9 transgene.
  • the plasmid contains NRF2-targeting gRNAs driven by U6, H1 and 7SK promoters followed by the NQO1 promoter region, shown in white, driving eSpCas9 expression.
  • Figure 10 Vector map of pAd-U6-R34G-NQO1pro-Cas9.
  • the NQO1 promoter region was ligated into an adenoviral plasmid preceding the Cas9 transgene.
  • the plasmid contains a single gRNA targeting the R34G mutation in NRF2 driven by a U6 promoter followed by the NQO1 promoter region, shown in white, driving eSpCas9 expression. 22 ME147020341v.2 130949-01420 [0057]
  • Figure 11. Vector map of pAd-U6-Neh2-3-NQO1pro-Cas9. The NQO1 promoter region was ligated into an adenoviral plasmid preceding the Cas9 transgene.
  • the plasmid contains a single gRNA targeting NRF2 driven by a U6 promoter followed by the NQO1 promoter region, shown in white, driving eSpCas9 expression.
  • FIG. 13 Schematic diagram of Tandem ARE-based vector. The diagram depicts five to ten ARE sequences from NQO1 along with a minimal promoter driving expression of Cas9 in a plasmid-based vector.
  • Figure 13 Schematic diagram of other ARE-based vector. The diagram depicts the promoter region of another ARE-containing target gene of NRF2 used to drive expression of Cas9 in a plasmid-based vector.
  • Figure 14 Schematic diagram of TRE-based vector. The diagram depicts the promoter region of another ARE-containing target gene of NRF2 used to drive expression of Cas9 in a plasmid-based vector.
  • Figure 15 Schematic Diagram of Tandem TRE-based vector.
  • FIG. 16 Schematic diagram of FOXA1 promoter-based vector. The diagram depicts the promoter region of FOXA1 to drive the expression of Cas9 in a plasmid-based vector.
  • Figure 17. Schematic diagram of pAd-U6-fLucgRNA44-NQO1-Cas9 and sequencing confirmation of fLuc gRNA 44 (SEQ ID NO:22).
  • Figure 18. Western blot analysis of NRF2-mutated H1703 cell lines with and without CRISPR targeting.
  • the cells were transfected with a scramble gRNA or NRF2 gRNA and collected for protein analysis of NRF2.
  • the scramble gRNA samples depict the basal levels of NRF2 present in the cells.
  • Figure 19 Luciferase Activity after transfection of pAd-U6-fLucgRNA44- NQO1pro-Cas9 and pAd-U6-fLucgRNA44-nopro-Cas9 vectors.
  • the graph displays raw luminescence values of each respective plasmid transfected by lipofection. Luciferase activity was determined 48 hours post transfection and displayed as a raw luminescent value.
  • 23 ME147020341v.2 130949-01420 [0066] Figure 20.
  • Lane 1 contains the PCR amplification of cDNA converted from RNA of cells that were not transfected with any plasmid DNA therefore there should be no amplification.
  • Lane 2 contains the PCR amplification of cDNA converted from RNA of cells transfected with pAd-U6-fLucgRNA44-NQO1-Cas9.
  • DETAILED DESCRIPTION Applicant has solved the problem and overcome the limitations discussed above through a novel approach using gene-specific transcriptional elements in combination with a promoter to drive CRISPR/Cas expression.
  • the term “about” or “approximately” means within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less of a given value or range.
  • the term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of”. Similarly, the term “consisting essentially of” is intended to include embodiments encompassed by the term “consisting of”.
  • a reference to “A and/or B”, when used in conjunction with open- ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as “and/or” as defined above.
  • an “endonuclease” an enzyme that cleaves the phosphodiester bond within a polynucleotide chain.
  • an endonuclease generates a double- stranded break at a desired position in the genome, and in some embodiments, an endonuclease generates a single-stranded break or a “nick” or break on one strand of the DNA phosphate sugar backbone at a desired position in the genome, and in some embodiments, without producing undesired off-target DNA stranded breaks.
  • Endonuclease can be naturally occurring endonuclease or it can be artificially generated.
  • a “Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)- associated endonuclease protein-binding domain” or “Cas binding domain” refers to a nucleic acid element or domain within a nucleic acid sequence or polynucleotide sequence that, in an effective amount, will bind or have an affinity for one or a plurality of CRISPR-associated endonuclease (or functional fragments thereof).
  • the one or plurality of proteins and the nucleic acid element forms a biologically active CRISPR complex and/or can be enzymatically active on a target sequence.
  • the CRISPR- associated endonuclease is a class 1 or class 2 CRISPR-associated endonuclease, and in some embodiments, a Cas9 or Cas12a endonuclease.
  • the Cas9 endonuclease can have a nucleotide sequence identical to the wild type Streptococcus pyogenes sequence.
  • the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as thermophilus; Pseudomonas aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms.
  • Such species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Alicycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammap
  • the CRISPR- associated endonuclease can be a Cas12a nuclease.
  • the Cas12a nuclease can have a nucleotide sequence identical to a wild type Prevotella, Francisella, Acidaminococcus, Proteocatella, Sulfurimonas, Elizabethkingia, Methylococcales, Moraxella, Helcococcus, Lachnospira, Limihaloglobus, Butyrivibrio, Methanomethylophilus, Coprococcus, Synergistes, Eubacterium, Roseburia, Bacteroidales, Ruminococcus, Eubacteriaceae, Leptospira, Parabacteriodes, Gracilibacteria, Lachnospiraceae, Clostridium, Brumimicrobium, Fibrobacter, Catenovulum, Acinetobacter, Flavobacterium, Succiniclasticum, Pseudobutyrivibrio, Barnes
  • the terms “(CRISPR)-associated endonuclease protein- binding domain” or “Cas binding domain” refer to a nucleic acid element or domain (e.g. and RNA element or domain) within a nucleic acid sequence that, in an effective amount, will bind to or have an affinity for one or a plurality of CRISPR-associated 27 ME147020341v.2 130949-01420 endonucleases (or functional fragments or variants thereof that are at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to a CRISPR-associated endonuclease).
  • the Cas binding domain consists of at least or no more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,
  • CRISPR CRISPR associated
  • Cas CRISPR-Cas system guide RNA
  • CRISPR-Cas system guide RNA may comprise a transcription terminator domain.
  • transcription terminator domain refers to a nucleic acid element or domain within a nucleic acid sequence (or polynucleotide sequence) that, in an effective amount, prevents bacterial transcription when the CRISPR complex is in a bacterial species and/or creates a secondary structure that stabilizes the association of the nucleic acid sequence to one or a plurality of Cas proteins (or functional fragments thereof) such that, in the presence of the one or a plurality of proteins (or functional fragments thereof), the one or plurality of Cas proteins and the nucleic acid element forms a biologically active CRISPR complex and/or can be enzymatically active on a target sequence in the presence of such a target sequence and a DNA-binding domain.
  • the transcription terminator domain consists of at least or no more than about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 28 ME147020341v.2 130949-01420 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190
  • DNA-binding domain refers to a nucleic acid element or domain within a nucleic acid sequence (e.g. a guide RNA) that is complementary to a target sequence.
  • the DNA-binding domain will bind or have an affinity for a target sequence such that, in the presence of a biologically active CRISPR complex, one or plurality of Cas proteins can be enzymatically active on the target sequence.
  • the DNA binding domain comprises at least one sequence that is capable of forming Watson Crick basepairs with a target sequence as part of a biologically active CRISPR system at a concentration and microenvironment suitable for CRISPR system formation.
  • CRISPR system refers collectively to transcripts or synthetically produced transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.
  • a tracr trans-activating CRISPR
  • tracr-mate sequence encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • guide sequence also referred to as a “spacer” in the context of an endogen
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a nucleic acid sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • the target sequence is a DNA polynucleotide and is referred to a DNA target sequence.
  • a target sequence comprises at least three nucleic acid sequences that are recognized by a Cas-protein when the Cas protein is associated with a CRISPR complex or system which comprises at least one sgRNA or one tracrRNA/crRNA duplex at a concentration and within an microenvironment suitable for association of such a system.
  • the target DNA comprises at least one or more proto-spacer adjacent motifs which sequences are known in the art and are dependent upon the Cas protein system being used in conjunction with the sgRNA or crRNA/tracrRNAs employed by this work.
  • the target DNA comprises NNG, where G is an guanine and N is any naturally occurring nucleic acid.
  • the target DNA comprises any one or combination of NNG, NNA, GAA, NGGNG, NGRRT, NGRRN, NNNNGATT, NNNNRYAC, NNAGAAW, TTTV, YG, TTTN, YTN, NGCG, NGAG, NGAN, NGNG, NG, NNGRRT, TYCV, TATV, or NAAAAC.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • a CRISPR complex comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins
  • formation of a CRISPR complex results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 30 ME147020341v.2 130949-01420 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more nucleotides of a wild-
  • the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional (bind the Cas protein or functional fragment thereof).
  • the tracr sequence has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that the presence and/or expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.
  • the guide sequence or RNA or DNA sequences that form a CRISPR complex are at least partially synthetic.
  • the CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element.
  • the disclosure relates to a composition comprising a chemically synthesized guide sequence.
  • the chemically synthesized guide sequence is used in conjunction with a 31 ME147020341v.2 130949-01420 vector comprising a coding sequence that encodes a CRISPR enzyme, such as a class 2 Cas9 or Cas12a protein.
  • the chemically synthesized guide sequence is used in conjunction with one or more vectors, wherein each vector comprises a coding sequence that encodes a CRISPR enzyme, such as a class 2 Cas9 or Cas12a protein.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more additional (second, third, fourth, etc.) guide sequences, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).
  • the CRISPR enzyme, one or more additional guide sequence, tracr mate sequence, and tracr sequence are each a component of different nucleic acid sequences.
  • the disclosure relates to a composition
  • a composition comprising at least a first and second nucleic acid sequence, wherein the first nucleic acid sequence comprises a tracr sequence and the second nucleic acid sequence comprises a tracr mate sequence, wherein the first nucleic acid sequence is at least partially complementary to the second nucleic acid sequence such that the first and second nucleic acid for a duplex and wherein the first nucleic acid and the second nucleic acid either individually or collectively comprise a DNA-targeting domain, a Cas protein binding domain, and a transcription terminator domain.
  • the CRISPR enzyme, one or more additional guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.
  • the disclosure relates to compositions comprising any one or combination of the disclosed domains on one guide sequence or two separate tracrRNA/crRNA sequences with or without any of the disclosed modifications. Any methods disclosed herein also relate to the use of tracrRNA/crRNA sequence interchangeably with the use of a guide sequence, such that a composition may 32 ME147020341v.2 130949-01420 comprise a single synthetic guide sequence and/or a synthetic tracrRNA/crRNA with any one or combination of modified domains disclosed herein.
  • a guide RNA can be a short, synthetic, chimeric tracrRNA/crRNA (a “single-guide RNA” or “sgRNA”).
  • a guide RNA may also comprise two short, synthetic tracrRNA/crRNAs (a “dual-guide RNA” or “dgRNA”).
  • dgRNA dual-guide RNA
  • the term “homologous” or “homologue” or “ortholog” refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity.
  • the terms “homology”, “homologous”, “substantially similar”, and “corresponding substantially” are used interchangeably herein.
  • nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype.
  • modifications of the nucleic acid fragments of the instant disclosure such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment.
  • these terms describe the relationship between a gene found in one species, subspecies, variety, cultivar, or strain and the corresponding or equivalent gene in another species, subspecies, variety, cultivar or strain. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.
  • double-strand break refers to the severing or cleavage of both strands of the DNA double helix.
  • the DSB may result in cleavage of both stands at the same position leading to “blunt ends” or staggered cleavage resulting in a region of single-stranded DNA at the end of each DNA 33 ME147020341v.2 130949-01420 fragment, or “sticky ends”.
  • a DSB may arise from the action of one or more DNA nucleases.
  • hybridizable By “hybridizable”, “complementary”, or “substantially complementary” it is meant that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize”, to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength.
  • a nucleic acid e.g., RNA, DNA
  • anneal i.e., antiparallel
  • Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C).
  • adenine (A) pairing with thymidine (T)
  • A adenine
  • U uracil
  • G guanine
  • C cytosine
  • RNA molecules e.g., dsRNA
  • guanine (G) can also base pair with uracil (U).
  • G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA.
  • a guanine (G) e.g., of a protein-binding segment (dsRNA duplex) of a subject guide nucleic acid molecule, of a target nucleic acid base pairing with a guide nucleic acid, and/or a PAMmer, etc.
  • G guanine
  • U uracil
  • A an adenine
  • a G/U base-pair can be made at a given nucleotide position of a protein-binding segment (e.g., dsRNA duplex) of a subject guide nucleic acid molecule
  • the position is not considered to be non- complementary, but is instead considered to be complementary.
  • Hybridization and washing conditions are well known and exemplified in Sambrook J., Fritsch. E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook. J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001).
  • Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible.
  • the conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (T m ) for hybrids of nucleic acids having those sequences.
  • the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8).
  • the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more).
  • the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, or 37 °C; hybridization buffer concentrations of about 6 ⁇ SSC, 7 ⁇ SSC, 8 ⁇ SSC, 9 ⁇ SSC, or 10 ⁇ SSC; formamide concentrations of about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%; and wash solutions from about 4 ⁇ SSC, 5 ⁇ SSC, 6 ⁇ SSC, 7 ⁇ SSC, to 8 ⁇ SSC.
  • moderate hybridization conditions include: incubation temperatures of about 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, or 50 °C; buffer concentrations of about 9 ⁇ SSC, 8 ⁇ SSC, 7 ⁇ SSC, 6 ⁇ SSC, 5 ⁇ SSC, 4 ⁇ SSC, 3 ⁇ SSC, or 2 ⁇ SSC; formamide concentrations of about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%; and wash solutions of about 5 ⁇ SSC, 4 ⁇ SSC, 3 ⁇ SSC, or 2 ⁇ SSC.
  • high stringency conditions include: incubation temperatures of about 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, or 68%; buffer concentrations of about 1 ⁇ SSC, 0.95 ⁇ SSC, 0.9 ⁇ SSC, 0.85 ⁇ SSC, 35 ME147020341v.2 130949-01420 0.8 ⁇ SSC, 0.75 ⁇ SSC, 0.7 ⁇ SSC, 0.65 ⁇ SSC, 0.6 ⁇ SSC, 0.55 ⁇ SSC, 0.5 ⁇ SSC, 0.45 ⁇ SSC, 0.4 ⁇ SSC, 0.35 ⁇ SSC, 0.3 ⁇ SSC, 0.25 ⁇ SSC, 0.2 ⁇ SSC, 0.15 ⁇ SSC, or 0.1 ⁇ SSC; formamide concentrations of about 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 7
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes or more. It is understood that equivalents of SSC using other buffer systems can be employed. [0092] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure).
  • a polynucleotide can comprise about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% (i.e., full complementarity) sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize.
  • an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize would represent 90% complementarity.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.
  • Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Exemplary methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol.
  • a “variant”, “mutant”, or “mutated” polynucleotide contains at least one polynucleotide sequence alteration as compared to the polynucleotide sequence of the corresponding wild-type or parent polynucleotide.
  • CRISPR/Endonucleases [0094] CRISPR/Endonucleases [0095] CRISPR/endonuclease (e.g., CRISPR/Cas9) systems are known in the art and are described, for example, in U.S. Patent No.9,925,248, which is incorporated by reference herein in its entirety. CRISPR-directed gene editing can identify and execute DNA cleavage at specific sites within the chromosome at a surprisingly high efficiency and precision. The natural activity of CRISPR/Cas9 is to disable a viral genome infecting a bacterial cell. Subsequent genetic reengineering of CRISPR/Cas function in human cells presents the possibility of disabling human genes at a significant frequency.
  • CRISPR/Endonucleases e.g., CRISPR/Cas9 systems are known in the art and are described, for example, in U.S. Patent No.9,925,248, which is incorporated by reference herein in
  • 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) containing a DNA binding region (spacer) which is complementary to the target gene.
  • the compositions described herein can include a nucleic acid encoding a CRISPR-associated endonuclease.
  • the CRISPR-associated endonuclease can be, e.g., a class 1 CRISPR-associated endonuclease or a class 2 CRISPR-associated endonuclease.
  • Class 1 CRISPR-associated endonucleases include type I, type III, and type IV CRISPR-Cas systems, which have effector molecules that comprise multiple subunits.
  • effector molecules can 37 ME147020341v.2 130949-01420 include, in some embodiments, Cas7 (csm3, cmr1, cmr6) and Cas5 (csm4, csx10, cmr3), along with, in some embodiments, SS (Cas11) and Cas8a1; Cas8b1; Cas8c; Cas8u2 and Cas6; Cas3" and Cas10d; SS (Cas11), Cas8e, and Cas6; Cas8f and Cas6f; Cas6f; Cas8-like (Csf1); SS (Cas11) and Cas8-like (Csf1); or SS (Cas11) and Cas10.
  • Class 1 CRISPR-associated endonucleases also be associated with, in some embodiments, target cleavage molecules, which can be Cas3 (type I) or Cas10 (type III) and spacer acquisition molecules such as, e.g., Cas1, Cas2, and/or Cas4.
  • target cleavage molecules which can be Cas3 (type I) or Cas10 (type III) and spacer acquisition molecules such as, e.g., Cas1, Cas2, and/or Cas4.
  • Class 2 CRISPR-associated endonucleases include type I, type V, and type VI CRISPR-Cas systems, which have a single effector molecule.
  • effector molecules can include, in some embodiments, Cas9, Cas12a (cpf1), Cas12b1 (c2c1), Cas12b2, Cas12c (c2c3), Cas12d (CasY), Cas12e (CasX), Cas12f1 (Cas14a), Cas12f2 (Cas14b), Cas12f3 (Cas14c), Cas12g, Cas12h, Cas12i, Cas12k (c2c5), Cas13a (c2c2), Cas13b1 (c2c6), Cas13b2 (c2c6), Cas13c (c2c7), Cas13d, c2c4, c2c8, c2c9
  • the CRISPR-associated endonuclease can be from Acetobacter pasteurianus, Acetobacter persici, Acetobacter sp., Acetobacterium woodii, Acetohalobium arabaticum, Acholeplasma palmae, Acidaminococcus fermentans, Acidaminococcus intesini, Acidihalobacter ferrooxidans, Acidimicrobium ferrooxidans, Acidiphilium cryptum, Acidipropionibacterium acidipropionici, Acidithiobacillus caldus, Acidobacterium capsulatum, Acidothermus celluloyticus, Acidovorax avenae, Acidovorax sp., Acinetobacter baumannii, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter soli, Acinetobacter sp., Actinoalloteichus hymeniacidonis, Actinoalloteichus s
  • Clostridium sacchrolyticum Clostridium chauvoei, Clostridium clariflavum, Clostridium difficile, Clostridium formicaceticum, Clostridium kluyveri, Clostridium novyi, Clostridium pasteurianum, Clostridium perfringens, Clostridium saccharobutylicum, Clostridiumscatologenes, Clostridium sp., Clostridium sporogenes, Clostridium stercorarium, Clostridium tetani, Clostridium thermocellum, Clostridium tyrobutyricum, Comamonadaceae bacterium, Comamonas kersterii, Confluentimicrobium sp., Coprococcus catus, Coprococcus sp., Coprothermobacter 40 ME147020341v.2 130949-01420 proteolyticus, Corallococcus coralloides, Coriobacteri
  • the CRISPR-associated endonuclease can be a Cas9 nuclease.
  • the Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyogenes sequence.
  • the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as thermophilus; Pseudomonas aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms.
  • Such species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Alicycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammap
  • the wild type Streptococcus pyogenes Cas9 sequence can be modified.
  • the nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, e.g., human cells.
  • a Cas9 nuclease sequence codon optimized for 51 ME147020341v.2 130949-01420 expression in human cells sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 GI:669193757; KM099232.1 GI:669193761; or KM099233.1 GI:669193765.
  • the Cas9 nuclease sequence can be, for example, the sequence contained within a commercially available vector such as pX458, pX330 or pX260 from Addgene (Cambridge, Mass.).
  • 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 GI:669193757; KM099232.1 GI:669193761; or KM099233.1 GI:669193765 or Cas9 amino acid sequence of pX458, pX330 or pX260 (Addgene, Cambridge, Mass.).
  • 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, e.g., insertions, deletions, or mutations or a combination thereof.
  • One or more of the 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%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a wild type Cas9 polypeptide.
  • sequence identity e.g., at least or about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
  • the CRISPR-associated endonuclease can be a Cas12a nuclease.
  • the Cas12a nuclease can have a nucleotide sequence identical to a wild type Prevotella or Francisella sequence. Alternatively, a wild type Prevotella or Francisella Cas12a sequence can be modified.
  • an Acidaminococcus Proteocatella, Sulfurimonas, Elizabethkingia, Methylococcales, Moraxella, Helcococcus, Lachnospira, Limihaloglobus, Butyrivibrio, Methanomethylophilus, Coprococcus, Synergistes, Eubacterium, Roseburia, Bacteroidales, Ruminococcus, Eubacteriaceae, Leptospira, Parabacteriodes, Gracilibacteria, Lachnospiraceae, Clostridium, Brumimicrobium, Fibrobacter, 52 ME147020341v.2 130949-01420 Catenovulum, Acinetobacter, Flavobacterium, Succiniclasticum, Pseudobutyrivibrio, Barnesiella, Sneathia, Succinivibrionaceae, Treponema, Sedimentisphaera, Thiomicrospira, Eucomonympha,
  • the nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, e.g., human cells.
  • a Cas12a nuclease sequence codon optimized for expression in human cells sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers MF193599.1 GI: 1214941796, KY985374.1 GI: 1242863785, KY985375.1 GI: 1242863787, or KY985376.1 GI: 1242863789.
  • the Cas12a nuclease sequence can be, for example, the sequence contained within a commercially available vector such as pAs-Cpf1 or pLb-Cpf1 from Addgene (Cambridge, Mass.).
  • the Cas12a endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas12a endonuclease sequences of Genbank accession numbers MF193599.1 GI: 1214941796, KY985374.1 GI: 1242863785, KY985375.1 GI: 1242863787, or KY985376.1 GI: 1242863789 or Cas12a amino acid sequence of pAs-Cpf1 or pLb-Cpf1 (Addgene, Cambridge, Mass.).
  • the Cas12a nucleotide sequence can be modified to encode biologically active variants of Cas12a, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas12a by virtue of containing one or more, e.g., insertions, deletions, or mutations or a combination thereof.
  • One or more of the mutations can be a substitution (e.g., a conservative amino acid substitution).
  • a biologically active variant of a Cas12a polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a wild type Cas12a polypeptide.
  • sequence identity e.g., at least or about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 6
  • the Cas9 enzyme comprises catalytically dead Cas or a homolog, an ortholog or mimic thereof.
  • Catalytically dead Cas enzymes include, but 53 ME147020341v.2 130949-01420 are not limited to, proteins or peptides which are capable of interaction with an sgRNA to target a site of interest.
  • the Cas protein may be mutated so that the nuclease activity is inactivated.
  • An inactivated Cas protein (with no endonuclease activity may be targeted to genes by gRNAs to silence gene expression through steric hindrance.
  • compositions described herein may also include sequence encoding a guide RNA (gRNA) comprising a DNA-binding domain that is complementary to a target domain in a target sequence, and a CRISPR-associated endonuclease protein- binding domain.
  • the guide RNA sequence can be a sense or anti-sense sequence.
  • the guide RNA sequence may include a PAM.
  • the sequence of the PAM can vary depending upon the specificity requirements of the CRISPR endonuclease used. In, e.g., the CRISPR-Cas system derived from S. pyogenes, the target DNA typically immediately precedes a 5'-NGG proto-spacer adjacent motif (PAM). Thus, for the S.
  • PAM 5'-NGG proto-spacer adjacent motif
  • the PAM sequence can be NGG.
  • Other Cas endonucleases may have different PAM specificities (e.g., NNG, NNA, GAA, NGGNG, NGRRT, NGRRN, NNNNGATT, NNNNRYAC, NNAGAAW, TTTV, YG, TTTN, YTN, NGCG, NGAG, NGAN, NGNG, NG, NNGRRT, TYCV, TATV, or NAAAAC).
  • 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.
  • the DNA-binding domain varies in length from about 20 to about 55 nucleotides, for example, 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 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, or about 55 nucleotides.
  • the Cas protein-binding domain is from about 30 to about 55 nucleotides in length, for example, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, or about 55 nucleotides.
  • the compositions comprise one or more nucleic acid (i.e. DNA) sequences encoding the guide RNA and the CRISPR endonuclease.
  • the CRISPR endonuclease can be encoded by the same nucleic acid or vector as the guide RNA sequence.
  • the CRISPR endonuclease can be encoded in a physically separate nucleic acid from the guide RNA sequence or in a separate vector.
  • the nucleic acid sequence encoding the guide RNA may comprise a DNA binding domain, a Cas protein binding domain, and a transcription terminator domain.
  • the nucleic acid encoding the guide RNA and/or the CRISPR endonuclease may be an isolated nucleic acid.
  • 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.
  • Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA.
  • PCR polymerase chain reaction
  • PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies also are available by which site- specific nucleotide sequence modifications can be introduced into a template nucleic acid.
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides e.g., >50-100 nucleotides
  • Target cells may be prokaryotic or eukaryotic cells.
  • cells can be fungal cells, plant cells, protist cells, or animal cells.
  • the cell is selected from the group consisting of an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, an invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.
  • an archaeal cell a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, an invertebrate cell, a vertebrate cell, a fish cell, a
  • Recombinant constructs are also provided herein and can be used to transform cells in order to express the CRISPR endonuclease and/or a guide RNA complementary to a target sequence.
  • a recombinant nucleic acid construct may comprise a nucleic acid encoding a CRISPR endonuclease and/or a guide RNA complementary to a target sequence, operably linked to a promoter suitable for expressing the CRISPR endonuclease and/or a guide RNA complementary to the target sequence in the cell.
  • the nucleic acid encoding a CRISPR endonuclease is operably linked to the same promoter as the nucleic acid encoding the guide RNA.
  • the nucleic acid encoding a CRISPR endonuclease and the nucleic acid encoding the guide RNA are operably linked to different promoters.
  • the promoter can be one or more pol III promoters, one or more pol II promoters, one or more pol I promoters, or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV), LTR promoter (optionally with the RSV enhancer), the 56 ME147020341v.2 130949-01420 cytomegalovirus (CMV) promoter (optionally with the CMV enhancer; see, e.g., Boshart et al., Cell 41:521-30 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
  • RSV Rous sarcoma virus
  • LTR optionally with the RSV enhancer
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • a pol I promoter includes, but is not limited to, the 47S pre-rRNA promoter.
  • one or more CRISPR endonucleases and one or more guide RNAs may be provided in combination in the form of ribonucleoprotein particles (RNPs).
  • RNPs ribonucleoprotein particles
  • An RNP complex can be introduced into a subject by means of, e.g., injection, electroporation, nanoparticles (including, e.g., lipid nanoparticles), vesicles, and/or with the assistance of cell-penetrating peptides.
  • an RNP complex may be formed by annealing an gRNA to a Cas protein, as described in, e.g., Schumann et al., Proc. Natl. Acad. Sci. USA 112:10437- 42 (2015), and Hultquist et al., Cell Rep.17:1438-52 (2016).
  • an RNP may comprise an anionic polymer added to a composition comprising a gRNA and a Cas protein under conditions described, e.g., in US20220017882, incorporated by reference herein in its entirety.
  • one or more CRISPR endonucleases and one or more guide RNAs may be delivered by a lipid nanoparticle (LNP).
  • LNP refers to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.
  • a nanoparticle may range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.
  • LNPs may be made from cationic, anionic, or neutral lipids.
  • Neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol, may be included in LNPs as “helper lipids” to enhance transfection activity and nanoparticle stability.
  • LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.
  • the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride can be used.
  • DOTMA can be formulated alone or combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine (DOPE) or other 57 ME147020341v.2 130949-01420 cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells.
  • DOPE dioleoylphosphatidyl-ethanolamine
  • Suitable cationic lipids include, but are not limited to, 5- carboxyspermylglycinedioctadecylamide, 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-1-propanaminium, 1,2-Dioleoyl-3- Dimethylammonium-Propane, 1,2-Dioleoyl-3-Trimethylammonium-Propane.
  • Contemplated cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3- aminopropane, 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane, 1,2-dilinoleyloxy-N,N- dimethyl-3-aminopropane, 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane, N-dioleyl- N,N-dimethylammonium chloride, N,N-distearyl-N,N-dimethylammonium bromide, N- (1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide, 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,ci- s-9,12- octadecadienoxy)propan
  • non-cationic lipids can be used.
  • non-cationic lipid refers to any neutral, zwitterionic, or anionic lipid.
  • anionic lipid refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.
  • Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), DOPE, palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), 58 ME147020341v.2 130949-01420 dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE),
  • DNA vectors containing nucleic acids such as those described herein also are also provided.
  • a “DNA 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 DNA 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.
  • DNA 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.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).
  • the DNA 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, Conn.) sequences typically are expressed as a fusion 59 ME147020341v.2 130949-01420 with the encoded polypeptide.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine e.g., c-myc, hemagglutinin
  • FlagTM tag Kodak, New Haven, Conn.
  • the DNA vector can also include a regulatory region.
  • the term “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.
  • the response element is an ARE, a cAMP response element, a B recognition element, an AhR-responsive element, a dioxin-responsive element, a xenobiotic-responsive element, a hypoxia-responsive element, an estrogen response element, an androgen response element, a serum response element, a retinoic acid response element, a peroxisome proliferator hormone response element, a metal-responsive element, a DNA damage response element, an IFN-stimulated response element, an ROR-response element, a glucocorticoid response element, a calcium-response element CaRE1, a p53 response element, a thyroid hormone response element, a growth hormone response element, a sterol response element, a polycomb response element, a Vitamin D response element, or a Rev response element.
  • the promoter is RNA polymerase I, RNA polymerase II, RNA polymerase III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, ⁇ - actin promoter, phosphoglycerol kinase (PGK) promoter, or EF1 ⁇ promoter.
  • the promoter is a minimal promoter, which as used herein, refers to minimal portion of a promoter required to properly initiate transcription.
  • a minimal promoter comprises, consists essentially of, or consists of a TATA sequence and/or an mRNA initiation sequence.
  • operably linked refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory 60 ME147020341v.2 130949-01420 DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
  • the two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent.
  • a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
  • 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 macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • viral vectors such as adenoviruses (“Ad”), adeno- associated viruses (AAV), and vesicular stomatitis virus (VSV) and retroviruses
  • Ad adenoviruses
  • Ad adeno-associated viruses
  • VSV vesicular stomatitis virus
  • retroviruses vesicular stomatitis virus
  • 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.
  • such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
  • Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • markers such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • Other vectors 61 ME147020341v.2 130949-01420 include those described by Chen et al., BioTechniques 34:167-71 (2003). A large variety of such vectors are known in the art and are generally available.
  • 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 viral vector comprises a strong eukaryotic promoter operably linked to the polynucleotide e.g., a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the recombinant viral vector can include one or more of the polynucleotides therein, in some embodiments about one polynucleotide.
  • use of between from about 0.1 ng to about 4000 ⁇ g will often be useful e.g., about 0.1 ng to about 3900 ⁇ g, about 0.1 ng to about 3800 ⁇ g, about 0.1 ng to about 3700 ⁇ g, about 0.1 ng to about 3600 ⁇ g, about 0.1 ng to about 3500 ⁇ g, about 0.1 ng to about 3400 ⁇ g, about 0.1 ng to about 3300 ⁇ g, about 0.1 ng to about 3200 ⁇ g, about 0.1 ng to about 3100 ⁇ g, about 0.1 ng to about 3000 ⁇ g, about 0.1 ng to about 2900 ⁇ g, about 0.1 ng to about 2800 ⁇ g, about 0.1 ng to about 2700 ⁇ g, about 0.1 ng to about 2600 ⁇ g, about 0.1 ng to about 2500 ⁇ g, about 0.1 ng to about 2
  • Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses.
  • One HIV-based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.
  • DNA viral vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see, e.g., Geller et al., J. Neurochem. 64:487-96 (1995); Lim et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ.
  • HSV herpes simplex I virus
  • the polynucleotides described here may also be used with a microdelivery vehicle such as cationic liposomes, adenoviral vectors, and exosomes.
  • a microdelivery vehicle such as cationic liposomes, adenoviral vectors, and exosomes.
  • exosomes may be used for delivery of a nucleic acid encoding a CRISPR endonuclease and/or guide RNA to a target cell, e.g. a cancer cell.
  • Exosomes are nanosized vesicles secreted by a variety of cells and are comprised of cellular membranes. Exosomes can attach to target cells by a range of surface adhesion proteins and vector ligands (tetraspanins, integrins, CD11b and CD18 receptors), and deliver their payload to target cells.
  • tetraspanins, integrins, CD11b and CD18 receptors Several studies indicate that exosomes have a specific cell tropism, according to their characteristics and origin, which can be used to target them to disease tissues and/or organs.
  • Another delivery method is to use single stranded DNA producing vectors which can produce the expressed products intracellularly. See, e.g., Chen et al., BioTechniques, 34:167-71 (2003). [0125] In some embodiments, introduction of the one or more DNA sequences encoding the two or more gRNAs and the nucleic acid sequence encoding the CRISPR-associated endonuclease results in no off-site mutagenesis.
  • introduction of the one or more DNA sequences encoding the two or 65 ME147020341v.2 130949-01420 more gRNAs and the nucleic acid sequence encoding the CRISPR-associated endonuclease results in off-site mutagenesis at a frequency of only about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.
  • the present disclosure relates to a DNA sequence comprising a polynucleotide encoding a Cas endonuclease operably linked to a response element that is activated by a polynucleotide targeted by a CRISPR/Cas system that includes the aforementioned encoded Cas endonuclease.
  • Expression of the Cas endonuclease when accompanied by other necessary elements for CRISPR/Cas activity, reduces expression of the targeted polynucleotide resulting in reduced response element activity and reduced expression of the Cas endonuclease.
  • the targeted polynucleotide and the CRISPR/Cas system act together in a feedback loop that has the targeted polynucleotide, when expressed at high levels, activating expression of the Cas endonuclease, which as part of a CRISPR/Cas system, then cleaves the targeted polynucleotide, resulting in reduced expression/activity of the targeted polynucleotide, which in turn reduces expression of the Cas endonuclease.
  • the target polynucleotide can be upstream or downstream of one or more target genome sites that share a pathway with the target polynucleotide.
  • target genome sites include a polynucleotide sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide.
  • target genome sites include a disease associated gene or polynucleotide.
  • a “disease-associated” gene or polynucleotide refers to any gene or polynucleotide that yields transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissue as compared with tissues or cells of a non-disease control.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the transcribed or translated products may be known or unknown, and may be at a normal or abnormal level.
  • the disease-associated gene or polynucleotide is associated with PI3K/AKT signaling, ERK/MAPK signaling, glucocorticoid receptor signaling, axonal guidance signaling, ephrin receptor signaling, actin cytoskeleton signaling, Huntington’s disease signaling, apoptosis signaling, B cell receptor signaling, leukocyte extravasation signaling, integrin signaling, acute phase response signaling, PTEN signaling, p53 signaling, aryl hydrocarbon receptor signaling, xenobiotic metabolism signaling, SAPK/JNK signaling, PPAr/RXR signaling, NF- ⁇ B signaling, neuregulin signaling, Wnt or beta catenin signaling, insulin receptor signaling, IL-6 signaling, hepatic cholestasis, IGF-1 signaling, NRF2-mediated oxidative stress response, hepatic signaling, fibrosis or hepatic
  • the target polynucleotide can be part of a feedback loop.
  • One exemplary feedback loop is the NRF2/KEAP1 pathway.
  • NRF2 acts as the primary line of cellular defense against ROS to maintain oxidative homeostasis by regulating the expression of a plethora of genes involved in ROS clearance and rewiring of cellular metabolism.
  • KEAP1 a ubiquitin ligase adaptor protein, is a negative regulator of NRF2, the key transcription factor that activates the cellular antioxidant response.
  • NRF2/KEAP1 pathway an increase in the intracellular concentration of glutathione, or mutations in KEAP1 or NRF2 (e.g., a loss- of-function mutation in KEAP1, or a gain-of-function mutation in NRF2) confer a dependence of tumors on reduced glutathione, the major endogenous antioxidant comprised of glycine, cysteine, and glutamine-derived glutamate.
  • KEAP1 or NRF2 confers of tumors on reduced glutathione, the major endogenous antioxidant comprised of glycine, cysteine, and glutamine-derived glutamate.
  • NRF2 pathway Activation of the NRF2 pathway is important in preventing human diseases, such as cancer, neurodegenerative disease, cardiovascular diseases, ischemia, diabetes, pulmonary fibrosis, and inflammatory diseases. Conversely, high constitutive levels of NRF2 occur in many tumors or cancer cell lines. Moreover, overexpression of NRF2 in cancer cells protects them from the cytotoxic effects of anticancer therapies, resulting in chemo- and/or radioresistance. [0131] When NRF2 is ubiquitinated, it is transported to the proteasome, where it is degraded and its components recycled. Under normal conditions NRF2 has a half-life of only 20 minutes.
  • Oxidative stress or electrophilic stress disrupts critical cysteine residues in KEAP1, disrupting the KEAP1-Cul3 ubiquitination system.
  • NRF2 When NRF2 is not ubiquitinated, it builds up in the cytoplasm, and translocates into the nucleus. In the nucleus, it combines (forms a heterodimer) with a small Maf protein and binds to the ARE in the upstream promoter region of many antioxidative genes, and initiates their transcription.
  • the DNA sequence further comprises region(s) encoding one or more gRNAs of the CRISPR/Cas system, the one or more gRNAs having a binding site on the target polynucleotide, wherein each gRNA is operably linked to one or more promoters.
  • the one or more promoters are RNA polymerase I, RNA polymerase II, RNA polymerase III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, ⁇ -actin promoter, phosphoglycerol kinase (PGK) promoter, EF1 ⁇ promoter, or a combination thereof.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter cytomegalovirus
  • dihydrofolate reductase promoter ⁇ -actin promoter
  • PGK phosphoglycerol kinase
  • the response element is an antioxidant response element, a cAMP response element, a B recognition element, an AhR-responsive element, a dioxin-responsive element, a xenobiotic-responsive element, a hypoxia- responsive element, an estrogen response element, an androgen response element, a serum response element, a retinoic acid response element, a peroxisome proliferator hormone response element, a metal-responsive element, a DNA damage response element, an IFN-stimulated response element, an ROR-response element, a 69 ME147020341v.2 130949-01420 glucocorticoid response element, a calcium-response element CaRE1, an antioxidant response element, a p53 response element, a thyroid hormone response element, a growth hormone response element, a sterol response element, a polycomb response element, a Vitamin D response element, or a Rev response element.
  • the target sequence is NRF2, EGFR, EIF1AX, GNA11, SF3B1, BAP1, PBRM1, ATM, SETD2, KDM6A, CUL3, MET, SMARCA4, U2AF1, RBM10, STK11, NF1, NF2, IDH1, IDH2, PTPN11, MAX, TCF12, HIST1H1E, LZTR1, KIT, RAC1, ARID2, BRD4, BRD7, BARF1, NRAS, RNF43, SMAD4, ARID1A, ARID1B, KRAS, APC, SMAD2, SMAD3, ACVR2A, GNAS, HRAS, STAG2, FGFR3, FGFR4, RHOA, CDKN1A, ERBB3, KANSL1, RB1, TP53, CDKN2A, CDKN2B, CDKN2C, KEAP1, CASP8, TGFBR2, HLA-B, MAPK1, NOTCH1, NOTCH2,
  • the target is Nuclear Factor Erythroid 2-Related Factor (NRF2, NFE2L2).
  • NRF2 is considered the master regulator of 100-200 target genes involved in cellular responses to oxidative/electrophilic stress. Targets include glutathione (GSH) mediators, antioxidants and genes controlling efflux pumps.(Hayden et al., Urol. Oncol. Semin. Orig. Investig.32:806-14 (2014)).
  • GSH glutathione
  • NRF2 is also known to regulate expression of genes involved in protein degradation and detoxification and is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1), a substrate adapter for the Cul3-dependent E3 ubiquitin ligase complex.
  • KEAP1 Kelch-like ECH-associated protein 1
  • Keap1 constantly targets NRF2 for ubiquitin-dependent degradation maintaining low expression of NRF2 on downstream target genes.
  • chemotherapy has been shown to activate transcriptional activity of the NRF2 target genes often triggering a cytoprotective response; enhanced expression of NRF2 occurs in response to 71 ME147020341v.2 130949-01420 environmental stress or detrimental growth conditions.
  • Other mechanisms that lead to NRF2 upregulation include mutations in KEAP1 or epigenetic changes of the promoter region.
  • the upregulation of NRF2 expression leads to an enhanced resistance of cancer cells to chemotherapeutic drugs, which by their very action induce an unfavorable environment for cell proliferation. Indeed, Hayden et al.
  • CRISPR/Cas9 By using CRISPR/Cas9, it is possible to target and knock out the variant NRF2 protein causing chemoresistance, while not disrupting the function of wildtype NRF2 protein (US20200370041, incorporated herein by reference in its entirety).
  • some embodiments are directed to reducing or, in some embodiments, eliminating expression of variant NRF2s found only in cancer cells and not in non-cancerous cells. These variants are commonly found within the Neh2 Domain of NRF2, which is known as the KEAP1 binding domain.
  • the wild-type NRF2 sequence (NM_006164.5) is shown in SEQ ID NO:1: 1 gattaccgag tgccggggag cccggaggag ccgccgacgc agccgcacc gccgccgccg 61 ccgccaccag agccgccctg tccgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcg 121 gggagcccca acacacggtc cacagctcat catgatggac ttggagctgc cgcggg 181 actcccgtcc cagcaggaca tggatttgat tgacatactt tggaggcaag atatagatct 241 tggagtaagt cgagaa
  • the variant NRF2 comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:2 substituting positions 205-227 of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:13 (tgactgaagtcaaatacttctgg) substituting positions 273-251 of the reverse complement of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:10 substituting positions 374-396 of SEQ ID NO:1, SEQ ID NO:11 substituting positions 728-7
  • the variant NRF2 comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:14 (cttactccaagatctatatctgg) substituting positions 249-227 of the reverse complement of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:5 substituting positions 230-252 of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:10 substituting positions 374-396 of SEQ ID NO:1, SEQ ID NO:11 substituting positions 728-706
  • the variant NRF2 comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:14 substituting positions 74 ME147020341v.2 130949-01420 249-227 of the reverse complement of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:13 substituting positions 273-251 of the reverse complement of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:10 substituting positions 374-396 of SEQ ID NO:1, SEQ ID NO:11 substituting positions 7
  • the variant NRF2 comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:2 substituting positions 205-227 of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:5 substituting positions 230-252 of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:15 (caactagatgaagagacaggtgg) substituting positions 374-396 of SEQ ID NO:1, SEQ ID NO:11 substituting positions 728-706 of the reverse complement of
  • the variant NRF2 comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:2 substituting positions 205-227 of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:12 substituting positions 273-251 of the reverse complement of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 75 ME147020341v.2 130949-01420 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:15 substituting positions 374-396 of SEQ ID NO:1, SEQ ID NO:11 substituting positions 728-706 of the reverse
  • the variant NRF2 comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:14 substituting positions 249-227 of the reverse complement of SEQ ID NO:1, SEQ ID NO:3 substituting positions 215-237 of SEQ ID NO:1, SEQ ID NO:4 substituting positions 224-246 of SEQ ID NO:1, SEQ ID NO:5 substituting positions 230-252 of SEQ ID NO:1, SEQ ID NO:6 substituting positions 385-363 of the reverse complement of SEQ ID NO:1, SEQ ID NO:7 substituting positions 398-376 of the reverse complement of SEQ ID NO:1, SEQ ID NO:8 substituting positions 366-388 of SEQ ID NO:1, SEQ ID NO:9 substituting positions 411-389 of the reverse complement of SEQ ID NO:1, SEQ ID NO:15 substituting positions 374-396 of SEQ ID NO:1, SEQ ID NO:11 substituting positions 728-706 of the reverse complement of SEQ ID NO:1, or SEQ ID
  • Some embodiments are directed to methods of treating a tumor comprising introducing into a cell of the tumor a vector comprising a first polynucleotide encoding a Cas endonuclease of a CRISPR/Cas system, the first polynucleotide operably linked to a response element that is activated by a second polynucleotide targeted by the CRISPR/Cas system, whereby expression of the CRISPR/Cas system in the cell reduces expression of the second polynucleotide resulting in reduced response element activity and reduced expression of the first polynucleotide.
  • the tumor is a solid tumor.
  • the cancer is a non-small cell lung cancer.
  • treatment with the chemotherapeutic agent is initiated at the same time as treatment with a pharmaceutical composition disclosed herein.
  • the treatment with the chemotherapeutic agent is initiated after the treatment with a pharmaceutical composition disclosed herein is initiated.
  • treatment with the chemotherapeutic agent is initiated at before the treatment with a pharmaceutical composition disclosed herein.
  • the pharmaceutical compositions of the present disclosure may be utilized for the treatment of cancer wherein the subject has failed at least one prior chemotherapeutic regimen.
  • the cancer is resistant to one or more chemotherapeutic agents.
  • the present disclosure provides methods of treating cancer in a subject, wherein the subject has failed at least one prior chemotherapeutic regimen for the cancer, comprising administering the pharmaceutical compositions as described herein to the subject in an amount sufficient to treat the cancer, thereby treating the cancer.
  • the pharmaceutical compositions described herein may also be utilized for inhibiting tumor cell growth in a subject wherein the subject has failed at least one prior chemotherapeutic regimen.
  • the present disclosure further provides methods of inhibiting tumor cell growth in a subject, e.g. wherein the subject has failed at least one prior chemotherapeutic regimen, comprising administering the pharmaceutical compositions described herein to the subject, such that tumor cell growth is inhibited.
  • the subject is a mammal, e.g.
  • the pharmaceutical compositions described herein may be administered to a subject in an amount sufficient to reduce proliferation of cancer cells relative to cancer cells that are not treated with the pharmaceutical composition.
  • the pharmaceutical composition may reduce cancer cell proliferation by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
  • the pharmaceutical composition is administered in an amount sufficient to reduce tumor growth relative to a tumor that is not treated with the pharmaceutical composition.
  • the pharmaceutical composition may reduce tumor growth by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%
  • administration of the pharmaceutical composition to the subject completely inhibits tumor growth.
  • administration of a pharmaceutical composition as described herein achieves at least stable disease, reduces tumor size, inhibits tumor growth and/or prolongs the survival time of a tumor-bearing subject as compared to an appropriate control.
  • this disclosure also relates to a method of treating tumors in a human or other animal, including a subject, who has failed at least one prior 78 ME147020341v.2 130949-01420 chemotherapeutic regimen, by administering to such human or animal an effective amount of a pharmaceutical composition described herein.
  • a therapeutically active amount of the pharmaceutical composition may vary according to factors such as the disease stage (e.g., stage I versus stage IV), age, sex, medical complications, and weight of the subject, and the ability of the pharmaceutical composition to elicit a desired response in the subject.
  • the dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, the dose may be administered by continuous infusion, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the methods further include a treatment regimen which includes any one of or a combination of surgery, radiation, chemotherapy, e.g., hormone therapy, antibody therapy, therapy with growth factors, cytokines, and anti- angiogenic therapy.
  • a treatment regimen which includes any one of or a combination of surgery, radiation, chemotherapy, e.g., hormone therapy, antibody therapy, therapy with growth factors, cytokines, and anti- angiogenic therapy.
  • Cancers for treatment using the methods of the disclosed herein include, for example, all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. In one embodiment, cancers for treatment using the methods of disclosed herein include melanomas, carcinomas and sarcomas.
  • the coenzyme Q10 compositions are used for treatment, of various types of solid tumors, for example breast cancer, bladder cancer, colon and rectal cancer, endometrial cancer, kidney (renal cell) cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, cervical cancer, liver cancer, stomach cancer, mouth and oral cancers, neuroblastoma, testicular cancer, uterine cancer, thyroid cancer, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and medulloblastoma, and vulvar cancer.
  • solid tumors include breast cancer, including 79 ME147020341v.2 130949-01420 triple negative breast cancer.
  • skin cancer includes melanoma, squamous cell carcinoma, cutaneous T-cell lymphoma (CTCL).
  • CTCL cutaneous T-cell lymphoma
  • the cancer includes leukemia.
  • the cancer is selected from the group consisting of lung cancer, melanoma, esophageal squamous cancer (ESC), head and neck squamous cell carcinoma (HNSCC), and breast cancer.
  • the cancer is lung cancer, e.g. non-small-cell lung cancer (NSCLC).
  • the NSCLC is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.
  • combinatorial drug strategies used to treat NSCLC several different combinatorial approaches are also being investigated for the treatment of cancers.
  • the use of an oncolytic virus that infects tumor cells has been found to enhance the activity of chemotherapy. Infection with myxoma virus combined with cisplatin or gemcitabine efficiently destroyed ovarian cancer cells at much lower dosages than needed without viral addition (Nounamo et al., Mol. Ther. Oncolytics 6:90-99 (2017)).
  • the pharmaceutical compositions described herein can be used in combination therapy with at least one additional anticancer agent, e.g., a chemotherapeutic agent.
  • chemotherapeutic agents generally belong to various classes including, for example: 1.
  • Topoisomerase II inhibitors such as the anthracyclines/anthracenediones, e.g., doxorubicin, epirubicin, idarubicin and nemorubicin, the anthraquinones, e.g., mitoxantrone and losoxantrone, and the podophillotoxines, e.g., etoposide and teniposide; 2.
  • cytotoxic antibiotics such as the anthracyclines/anthracenediones, e.g., doxorubicin, epirubicin, idarubicin and nemorubicin, the anthraquinones, e.g., mitoxantrone and losoxantrone, and the podophillotoxines, e.g., etoposide and teniposide; 2.
  • mitotic inhibitors such as plant alkaloids (e.g., a compound belonging to a 80 ME147020341v.2 130949-01420 family of alkaline, nitrogen-containing molecules derived from plants that are biologically active and cytotoxic), e.g., taxanes, e.g., paclitaxel and docetaxel, and the vinka alkaloids, e.g., vinblastine, vincristine, and vinorelbine, and derivatives of podophyllotoxin; 3.
  • plant alkaloids e.g., a compound belonging to a 80 ME147020341v.2 130949-01420 family of alkaline, nitrogen-containing molecules derived from plants that are biologically active and cytotoxic
  • taxanes e.g., paclitaxel and docetaxel
  • vinka alkaloids e.g., vinblastine, vincristine, and vinorelbine, and derivatives of podophyllotoxin
  • Alkylating agents such as nitrogen mustards, ethyleneimine compounds, alkyl sulphonates and other compounds with an alkylating action such as nitrosoureas, dacarbazine, cyclophosphamide, ifosfamide and melphalan; 4.
  • Antimetabolites for example, folates, e.g., folic acid, fiuropyrimidines, purine or pyrimidine analogues such as 5-fluorouracil, capecitabine, gemcitabine, methotrexate, and edatrexate; 5.
  • Topoisomerase I inhibitors such as topotecan, irinotecan, and 9-nitrocamptothecin, camptothecin derivatives, and retinoic acid; and 6.
  • Platinum compounds/complexes such as cisplatin, oxaliplatin, and carboplatin.
  • chemotherapeutic agents for use in the methods of disclosed herein include, but are not limited to, amifostine (ethyol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carrnustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), doxorubicin lipo (doxil), gemcitabine (gemzar), daunorubicin, daunorubicin lipo (daunoxome), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine
  • the chemotherapeutic agent is selected from the group consisting of cisplatin, vinorelbine, carboplatin, and combinations thereof (e.g., cisplatin and vinorelbine; cisplatin and carboplatin; vinorelbine and carboplatin; cisplatin, vinorelbine, and carboplatin).
  • the pharmaceutical composition is administered in an amount sufficient to reduce tumor growth relative to a tumor that is treated with the at least one chemotherapeutic agent but is not treated with the pharmaceutical composition.
  • the pharmaceutical composition may reduce tumor growth by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8
  • Applicant specifically incorporates the entire contents of all cited references in this disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range or a list of upper values and lower values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or value and any lower range limit or value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the present disclosure be limited to the specific values recited when defining a range.
  • Example 1 Concept of CompleteKill, NRF2-driven expression of SpCas9 by ARE-dependent transcription
  • NRF2 Master transcription factor, NRF2, regulates expression of over 200 genes involved in oxidative and cytotoxic stresses. In normal cells, the level of NRF2 is tightly controlled by KEAP1 protein through ubiquitin-proteasome based proteolysis in cytoplasm.
  • NRF2 Under oxidative stress, KEAP1 no longer binds to NRF2, and NRF2 translocates to the nucleus of the cell where it forms heterodimers with other proteins and binds to the antioxidant response element (ARE) within target gene promoter regions. ARE binding by NRF2 recruits and initiates transcription by RNA polymerase II of the downstream gene ( Figure 1).
  • Another mechanism of activating NRF2 includes mutations either in NRF2 or KEAP1, which prevents the interaction between NRF2 and KEAP1. In cancer cells, such mutations have been shown to be prevalent, especially under chemotherapy treatment.
  • Applicant’s CompleteKill system utilizes NRF2-regulated promoters to express CRISPR/Cas nuclease to target genes of interest including NRF2 ( Figure 2).
  • the strength of promoter-specific luciferase expression was compared by cloning in a chicken beta actin promoter to drive luciferase expression This was conducted in the human lung adenocarcinoma cell line - A549, which has been characterized as having high levels of NRF2.
  • the NQO1 promoter region was cloned into an adenoviral plasmid to replace a chicken beta actin (CAG) promoter driving eSpCas9 expression ( Figure 3).
  • the adenoviral plasmid also contains NRF2 targeting gRNAs driven by U6, H1, and 7SK promoters as an all-in-one adenoviral construct.
  • NRF2 targeting gRNAs driven by U6, H1, and 7SK promoters as an all-in-one adenoviral construct.
  • high expression and activity of NRF2 like in many different cancer types, will initiate ARE-dependent transcription of the Cas9 transgene and allow for 83 ME147020341v.2 130949-01420 ribonucleoprotein complex formation with NRF2-targeting sgRNAs.
  • Formed NRF2- targeting CRISPR/Cas9 complexes will bind and cleave the NRF2 gene resulting in frame-shifting indels causing knockdown or knockout of the NRF2 protein.
  • Example 2 pGL3-NQO1pro construction
  • Applicant selected a 1118 bp region of the NQO1 promoter ( Figure 4. Genomic structure of NQO1). Transcription of NQO1 is directly activated by NRF2 through the ARE as part of the cytoprotective pathway within the cell, therefore NQO1 expression can be correlated to NRF2 expression.
  • the transcriptional activity of NRF2 through ARE binding was initially assessed using a luciferase backbone plasmid with the NQO1 promoter region cloned in to drive luciferase expression.
  • the NQO1 promoter was PCR amplified from genomic DNA obtained from the human-derived H1703 lung squamous cell carcinoma cell line.
  • the forward primer (TTTAAGCTTGACGGAGTCTCACTTCGT; SEQ ID NO:16) included a 5’ HindIII restriction site and the reverse primer (ACCGACCATGGCTCTGGTGCAGTC; SEQ ID NO:17) included a 5’ NcoI restriction site within the sequence of the primers.
  • the 1118bp NQO1 PCR amplicon was ligated into a blunt end TOPO vector and transformed into chemically competent E. coli cells. Several individual bacterial colonies were further cultured and miniprepped for Sanger sequencing to verify the sequence of the NQO1 promoter.
  • the TOPO vector containing the NQO1 promoter (TOPO-NQO1) was digested using HindIII and NcoI, run out on an agarose gel, and the 1114 bp NQO1 promoter fragment was gel extracted and purified for ligation.
  • the pGL3-Basic vector (Promega) was digested with HindIII and NcoI, checked by agarose gel and gel extracted to provide the backbone for NQO1 promoter-driven luciferase expression.
  • the 1114 bp NQO1 promoter region was ligated with the pGL3-basic vector backbone to create the final pGL3-NQO1pro vector as shown in Figure 5 (Vector map of pGL3-NQO1pro).
  • Example 3 pGL3-CAGpro construction
  • Applicant constructed a plasmid with chicken beta-actin (CAG) promoter-driven luciferase as a positive control.
  • the basic pGL3 luciferase reporter vector was purchased from Promega as above.
  • the chicken beta-actin (CAG) promoter was digested from plasmid DNA containing the promoter region.
  • the digested CAG fragment was ligated into a blunt-end TOPO vector and transformed into chemically competent E. coli cells.
  • Several individual bacterial colonies were further cultured and miniprepped for Sanger sequencing to verify the sequence of the CAG promoter.
  • the TOPO vector containing the CAG promoter was then digested using KpnI and BsaI restriction enzymes, run out on an agarose gel and the 812 bp CAG promoter fragment was gel-extracted and purified for ligation.
  • the pGL3-Basic vector was digested with KpnI and BsaI, checked by agarose gel and gel-extracted to provide the backbone for CAG promoter-driven luciferase expression.
  • the 812 bp CAG promoter region was ligated with the pGL3-Basic vector backbone to create the final pGL3-CAGpro vector as shown in Figure 6 (Vector map of pGL3-CAGpro).
  • Example 4 Promoter strength testing of pGL3-NQO1pro and pGL3- CAGpro [0169]
  • Applicant assessed the strength of NQO1 promoter by measuring luciferase activity based on pGL3-NQO1pro.
  • Human-derived A549 lung adenocarcinoma cells were transfected by nucleofection and lipofection with plasmids pGL3-NQO1pro, pGL3- CAGpro, and pGL3-control (Promega). Luciferase expression was measured at 48 and 72 hours post transfection by luminescence using the Steady-GLO assay (Promega).
  • a total of 2.5e5 cells were electroporated with 2.5 ⁇ g of pDNA per vector using the Lonza SF kit.1e4 cells were plated per well for a total of 6 replicates per condition per 96-well plate.
  • 1e4 cells were first plated per well for a total of 6 replicates per condition per 96-well plate. Twenty-fours after plating, Lipofectamine 3000 containing 200 ng of each pDNA vector was added per well for each condition per 96-well plate.
  • Steady-GLO reagent was added to each well and incubated for at least 5 mins before measuring 85 ME147020341v.2 130949-01420 luminescence using a Tecan Infinite 200 Pro plate reader.
  • the cell line A549 contains mutations in KEAP1 gene which results in activation of NRF2.
  • Applicant expected an expression of luciferase through the NQO1 promoter where activated NRF2 turns on the transcriptional activity by binding to the ARE elements.
  • a NRF2-targeting CRISPR/Cas9-expressing adenoviral plasmid (pAd-U6H17SK-R34G-CAGpro-Cas9) was used as the backbone for vector construction.
  • the plasmid was reconstructed to remove the CAG promoter.
  • the pAd-U6H17SK-R34G-CAGpro-Cas9 was digested with NcoI restriction enzyme, run out on an agarose gel, and the vector backbone (7412 bp) was gel-extracted and purified for further ligation.
  • the digested vector backbone was ligated with a synthesized fragment containing the 7SK promoter with gRNA region and gRNA scaffold region along with a filler sequencing containing a HindIII and NcoI restriction enzyme site.
  • This plasmid lacks a promoter to drive SpCas9 transgene expression thus termed “pAd-U6H17SK-R34G-nopro-Cas9” ( Figure 8. Vector map of pAd-U6H17SK- R34G-nopro-Cas9).
  • Example 6 pAd-U6H17SK-R34G-NQO1pro-Cas9 construction
  • the pAd-U6H17SK-R34G-nopro-Cas9 vector from Example 5 was used as the backbone for NQO1 promoter insertion.
  • the plasmid was digested with HindIII and NcoI restriction ezymes, run out on an agarose gel and the linearized vector backbone was gel-extracted and purified.
  • the TOPO-NQO1 vector and NQO1 promoter fragment from Example 1 were 86 ME147020341v.2 130949-01420 used to create the pAd-U6H17SK-R34G-NQO1pro-Cas9 vector.
  • the NQO1 promoter fragment was ligated with linearized pAd-U6H17SK-R34G-nopro-Cas9, transformed into chemically competent E. coli cells.
  • Several individual bacterial colonies were further cultured and miniprepped for Sanger sequencing to verify the sequence of the final vector (Figure 9. Vector map of pAd-U6H17SK-R34G-NQO1pro-Cas9).
  • Example 7 pAd-U6-R34G-NQO1pro-Cas9 construction
  • the pAd-U6H17SK- R34G-NQO1pro-Cas9 vector was digested with BglII and run out on an agarose gel.
  • the 6685 bp vector backbone and 1567 bp NQO1 promoter fragment was gel- extracted, purified and ligated together creating the pAd-U6-R34G-NQO1pro-Cas9 vector.
  • the vector was transformed into chemically competent E. coli cells.
  • FIG. 10 Vector map of pAd- U6-R34G-NQO1pro-Cas9).
  • Example 8 pAd-U6-Neh2-3-NQO1pro-Cas9 construction
  • the pAd-U6-R34G-NQO1pro-Cas9 vector was used as the starting plasmid for creating the pAd-U6-Neh2-3-NQO1pro-Cas9 vector.
  • the R34G 20 nt gRNA sequence (GATATAGATCTTGGAGTAAG; SEQ ID NO:18) was substituted with a new sequence Neh2 gRNA 3 sequence (GTGGAGGCAAGATATAGATCT; SEQ ID NO:19).
  • This sequence substitution was completed using the NEB Q5 Site-directed mutagenesis kit and web tool for primer design. The site-directed mutagenesis protocol was followed using the manufacturer’s recommendation.
  • the product was transformed into chemically competent E. coli cells. Several individual bacterial colonies were further cultured and miniprepped for Sanger sequencing to verify the sequence of the final vector (Figure 11. Vector map of pAd- U6-Neh2-3-NQO1pro-Cas9.
  • Example 9 (Prophetic) Assessment of NQO1 promoter driven Cas9 expression through the indel efficiency at the target site [0179] To assess activity and efficiency of NQO1 promoter-driven Cas9 expression, NRF2-expressing cells are transfected with pAd-U6H17SK-R34G-NQO1pro-Cas9, pAd-U6-R34G-NQO1pro-Cas9 or pAd-U6-Neh2-3-NQO1pro-Cas9 vectors. Forty-eight to 72 hours post transfection, cells are collected and genomic DNA is isolated.
  • genomic DNA is isolated, the region of interest – targeted by CRISPR/Cas9, is PCR amplified, and the PCR amplicon is used for further assessment by Sanger sequencing or Next-generation sequencing. Sequencing is assessed for indel efficiency at CRISPR the target site. Applicants expect to see a range of frameshifting and non-frameshifting deletions and insertions created at the target site by non-homologous end joining as a result of CRISPR-induced double stranded breaks. This result will lead to the protein knockout of NRF2 which in turn will decrease expression of Cas9.
  • Example 10 (Prophetic) Tandem AREs with minimal promoter construction
  • the pAd-U6H17SK-R34G-nopro-Cas9 vector is used as the backbone.
  • a tandem of five to ten ARE sequences from NQO1 are synthesized or constructed through cloning along with the minimal promoter fragment from chicken beta actin or CMV. Once the fragment is created, it is ligated into the plasmid backbone following the sgRNA transgene and before the Cas9 transgene, creating the pAd-U6H17SK-R34G-AREminpro-Cas9 vector.
  • Example 11 (Prophetic) Assessment of Tandem AREs with minimal promoter driven Cas9 expression through the indel efficiency at the target site 88 ME147020341v.2 130949-01420 [0183] To assess activity and efficiency of Tandem ARE-driven Cas9 expression, high NRF2-expressing cells are transfected with pAd-U6H17SK-R34G-AREminpro-Cas9 vector. Forty-eight to 72 hours post transfection, cells are collected, and genomic DNA is isolated.
  • genomic DNA is isolated, the region of interest – targeted by CRISPR/Cas9, is PCR amplified and the PCR amplicon is used for further assessment by Sanger sequencing or Next-generation sequencing. Sequencing is assessed for indel efficiency at the CRISPR target site. Applicant expects to see a range of frameshifting and non-frameshifting deletions and insertions created at the target site by non-homologous end joining as a result of CRISPR-induced double stranded breaks.
  • the genomic knockout of NRF2 leads to the protein knockout of NRF2 which in turn decreases the expression of Cas9.
  • Example 12 (Prophetic) other ARE containing promoter construction
  • the pAd-U6H17SK-R34G-nopro-Cas9 vector is used as the backbone.
  • the promoter region containing the ARE sequence from another gene such as GSTA or HMOX1 is PCR amplified from genomic DNA.
  • the primers, forward and reverse respectively, include 5' HindIII restriction site and a 5' NcoI restriction site within the sequence of the primers.
  • the PCR amplicon is ligated into a blunt end TOPO vector and transformed into chemically competent E. coli cells.
  • the TOPO vector containing the promoter is digested using HindIII and NcoI, run out on an agarose gel and the promoter fragment is gel extracted and purified for ligation. Once purified, it is ligated into the pAd-U6H17SK-R34G-nopro- Cas9 plasmid backbone following the sgRNA transgene and before the Cas9 transgene, creating the pAd-U6H17SK-R34G-AREpro-Cas9 vector. The vector is transformed into chemically competent E. coli cells.
  • Example 13 (Prophetic) Assessment of other ARE containing promoter driven Cas9 expression through the indel efficiency at the target site [0187] To assess activity and efficiency of other ARE-driven Cas9 expression, high NRF2-expressing cells are transfected with pAd-U6H17SK-R34G-AREpro-Cas9 vector. Forty-eight to 72 hours post transfection, cells are collected and genomic DNA is isolated.
  • genomic DNA is isolated, the region of interest – targeted by CRISPR/Cas9, is PCR amplified and the PCR amplicon is used for further assessment by Sanger sequencing or Next-generation sequencing. Sequencing is assessed for indel efficiency at the CRISPR target site. Applicant expects to see a range of frameshifting and non-frameshifting deletions and insertions created at the target site by non-homologous end joining as a result of CRISPR-induced double stranded breaks.
  • the genomic knockout of NRF2 leads to the protein knockout of NRF2 which in turn decreases the expression of Cas9.
  • Example 14 (Prophetic) KRAS-driven expression of SpCas9 by TRE- dependent transcription targeting KRAS
  • Oncogenic KRAS mutations occur in approximately 30% of all cancer types and most result in impaired GTPase activity causing constitutive activation of RAS signaling.
  • the activation of RAS signaling has been reported to cause resistance to anti-cancer drugs, in large part attributed to the upregulation or overexpression of NRF2 and its downstream pathway (Tao et al.2014).
  • Mutant KRAS has been identified to influence transcriptional regulation of NRF2 through a TPA-responsive element (TRE motif – TGCGTCA) within the promoter of NRF2. This transcriptional activity can be exploited.
  • mutant KRAS found in many different cancer types, will initiate TRE-dependent transcription of the Cas9 transgene and allow for ribonucleoprotein complex formation with KRAS-targeting sgRNAs.
  • KRAS-targeting CRISPR/Cas9 complexes will bind and cleave the KRAS gene resulting in frame-shifting indels causing knockdown or knockout of the KRAS protein.
  • 90 ME147020341v.2 130949-01420 Reduction of KRAS expression due to CRISPR-directed knockout in turn reduces transcription and expression of the Cas9 transgene.
  • Example 15 (Prophetic) TRE-based vector construction
  • the pAd-KRASgRNA-nopro-Cas9 vector is used as the backbone.
  • the promoter region containing the TRE sequence from NRF2 (+227-+542) is PCR amplified from genomic DNA.
  • the primers, forward and reverse respectively, include 5' HindIII restriction site and a 5' NcoI restriction site within the sequence of the primers.
  • the PCR amplicon is ligated into a blunt end TOPO vector and transformed into chemically competent E. coli cells.
  • the TOPO vector containing the promoter is digested using HindIII and NcoI, run out on an agarose gel and the promoter fragment is gel extracted and purified for ligation. Once purified, it is ligated into the pAd-KRASgRNA-nopro-Cas9 plasmid backbone following the sgRNA transgene and before the Cas9 transgene, creating the pAd-KRASgRNA-NRF2pro-Cas9 vector. The vector is transformed into chemically competent E. coli cells.
  • Example 16 Assessment of NRF2 promoter driven Cas9 expression through the indel efficiency at the target site [0193] To assess activity and efficiency of NRF2 promoter-driven Cas9 expression, high KRAS expressing cells are transfected with pAd-KRASgRNA-NRF2pro-Cas9 vectors. Forty-eight to 72 hours post transfection, cells are collected and genomic DNA is isolated.
  • genomic DNA is isolated, the region of interest – targeted by CRISPR/Cas9, is PCR amplified and the PCR amplicon is used for further assessment by Sanger sequencing or Next-generation sequencing. Sequencing is assessed for indel efficiency at CRISPR the target site. Applicant expects to see a range of 91 ME147020341v.2 130949-01420 frameshifting and non-frameshifting deletions and insertions created at the target site by non-homologous end joining as a result of CRISPR-induced double stranded breaks. This result will lead to the protein knockout of KRAS which in turn will decrease expression of Cas9.
  • Example 17 (Prophetic) Tandem TREs with minimal promoter construction
  • the pAd-KRASgRNA-nopro-Cas9 vector is used as the backbone.
  • Five to ten TRE sequences from NRF2 are synthesized or constructed through cloning along with the minimal promoter fragment from chicken beta actin or CMV. Once the fragment is created, it is ligated into the plasmid backbone following the sgRNA transgene and before the Cas9 transgene, creating the pAd-KRASgRNA- TREminpro-Cas9 vector.
  • the vector is transformed into chemically competent E.
  • Example 18 (Prophetic) Assessment of Tandem AREs with minimal promoter driven Cas9 expression through the indel efficiency at the target site [0197] To assess activity and efficiency of Tandem ARE-driven Cas9 expression, high KRAS-expressing cells are transfected with pAd-KRASgRNA-TREminpro-Cas9 vector. Forty-eight to 72 hours post transfection, cells are collected and genomic DNA is isolated.
  • genomic DNA is isolated, the region of interest – targeted by CRISPR/Cas9, is PCR amplified and the PCR amplicon is used for further assessment by Sanger sequencing or Next-generation sequencing. Sequencing is assessed for indel efficiency at the CRISPR target site. Applicant expects to see a range of frameshifting and non-frameshifting deletions and insertions created at the target site by non-homologous end joining as a result of CRISPR-induced double stranded 92 ME147020341v.2 130949-01420 breaks.
  • the genomic knockout of KRAS leads to the protein knockout of KRAS which in turn decreases the expression of Cas9.
  • Example 19 (Prophetic) KRAS-driven expression of SpCas9 by TRE- dependent transcription targeting NRF2 [0199] Mutant KRAS has been identified to influence transcriptional regulation of NRF2 through a TPA-responsive element within the promoter of NRF2. This transcriptional activity can be exploited. Theoretically, constitutive activity of mutant KRAS, found in many different cancer types, will initiate TRE-dependent transcription of the Cas9 transgene and allow for ribonucleoprotein complex formation with NRF2-targeting sgRNAs. Targeting NRF2 by CRISPR/Cas9 regulated by KRAS, will decrease expression of NRF2 which will increase chemosensitivity in KRAS-mutated cancers.
  • Example 20 (Prophetic) FOXA1-driven expression of SpCas9 by TP53- dependent transcription
  • Mutant TP53 has been identified to influence transcriptional regulation of FOXA1 through binding within the FOXA1 promoter (Kim et al.2021). This transcriptional activity can be exploited. Theoretically, constitutive activity of mutant TP53, found in many cancer types, will initiate FOXA1 promoter-driven transcription of the Cas9 transgene and allow for the ribonucleprotein complex formation with TP53- targeting sgRNAs. Targeting TP53 by CRISPR/Cas9 will decrease expression of mutant TP53 which in will in turn decrease Cas9 expression.
  • Example 21 (Prophetic) FOXA1 promoter-based vector construction
  • the pAd-p53gRNA-nopro-Cas9 vector is used as the backbone.
  • the FOXA1 promoter region (-1355 to +263) is PCR amplified from genomic DNA.
  • the primers, forward and reverse respectively, include 5' HindIII restriction site and a 5' NcoI restriction site within the sequence of the primers.
  • the PCR amplicon is ligated into a blunt end TOPO vector and transformed into chemically competent E. coli cells.
  • the TOPO vector containing the promoter is digested using HindIII and NcoI, run out on an agarose gel and the promoter fragment is gel extracted and purified for ligation. Once purified, it is ligated into the pAd-p53gRNA-nopro-Cas9 plasmid backbone following the sgRNA transgene and before the Cas9 transgene, creating the pAd-p53gRNA-FOXA1pro-Cas9 vector. The vector is transformed into chemically competent E. coli cells.
  • Example 22 (Prophetic) Assessment of FOXA1 promoter-driven Cas9 expression through the indel efficiency at the target site
  • mutant TP53 expressing cells are transfected with pAd-p53gRNA-FOXA1pro-Cas9 vector. Forty-eight to 72 hours post transfection, cells are collected and genomic DNA is isolated.
  • genomic DNA is isolated, the region of interest – targeted by CRISPR/Cas9, is PCR amplified and the PCR amplicon is used for further assessment by Sanger sequencing or Next-generation sequencing. Sequencing is assessed for indel efficiency at the CRISPR target site. Applicant expects to see a range of frameshifting and non-frameshifting deletions and insertions created at the target site by non-homologous end joining as a result of CRISPR-induced double stranded breaks.
  • the genomic knockout of TP53 leads to the protein knockout of TP53 which in turn decreases the expression of Cas9.
  • Example 23 Application of CompleteKill in other plasmid-based technologies
  • CompleteKill has the potential to be applied to any technology delivering plasmid-based vectors such as AAV, adenovirus, or nanoplasmids (Lu et al.2017; Vermeire et al.2021; Suschak et al.2020).
  • Example 24 pAd-U6-fLucgRNA44-NQO1-Cas9 construction and testing [0209] To establish the ability of the NQO1 promoter to drive Cas9 expression, the pAd-U6-fLucgRNA44-NQO1-Cas9 was constructed to target a nonessential gene to test gene editing capabilities.
  • the fLuc gRNA 44 (GACCTGGCAGATGGAACCTCT, SEQ ID NO:20) was cloned the pAd-U6-R34GgRNA-NQO1-Cas9 backbone ( Figure 17).
  • the H170344-25 cell line was lentivirally transduced to contain the firefly luciferase gene for assessing gene editing activity (H170344-25-LV-fLuc).
  • the H1703 44-25 cell line contains an R34G mutation in the NRF2 gene, known to increase NRF2 protein stability and expression ( Figure 18).
  • the H170344-25-LV-fLuc cell line was transfected by lipofectamine 3000 following manufacturer’s protocol with the pAd-U6- fLucgRNA44-NQO1-Cas9 plasmid DNA. Cells were collected for downstream analysis by luciferase assay to assess knockdown of luciferase expression post gene editing as well as next generation sequencing to assess the indel efficiency.
  • Figure 19 depicts the knockdown of luciferase expression in cells transfected with pAd-U6-fLucgRNA44- NQO1-Cas9 as opposed to cells transfected with pAd-U6-fLucgRNA44-nopro-Cas9 containing no promoter driving Cas9 expression, therefore an experimental control.
  • Next generation sequencing results were assessed by CRISPresso to identify the distribution of indels and frequency of reads as shown in Figure 20.
  • the bulk population of cells treated with pAd-U6-fLucgRNA44-NQO1-Cas9 shows about 1.34% editing (Figure 20).
  • the pAd-U6-fLucgRNA44-NQO1-Cas9 transfected samples did in fact contain Cas9 cDNA that was able to be amplified as opposed to no amplification in control cells ( Figure 21).
  • Example 25 (Prophetic) Assessment of ARE-containing promoter expression of CRISPR/Cas9 in a plasmid backbone 95 ME147020341v.2 130949-01420 [0211] To assess the ability of an ARE-containing promoter to drive CRISPR/Cas9 expression in the presence of NRF2, various cell models with various levels of NRF2 protein expression are used (Table 1). Cells are collected 48-72 hours post transfection to assess the ARE-driven expression of Cas9 through qPCR to assess the expression of Cas9 mRNA as well as gene editing efficiency through genomic DNA sequencing. In the presence of high NRF2 expression, there should be high Cas9 expression through ARE-driven transcription and high editing efficiency as a result.
  • Example 26 (Prophetic) Assessment of ARE-containing promoter expression of CRISPR/Cas9 upon stimulation of NRF2 expression 96 ME147020341v.2 130949-01420 [0214] To test the ability of NRF2 expression to activate transcription in an environmental dependent manner, cells will be exposed to various NRF2 inducing or inhibiting stimuli to assess presence or absence of Cas9 expression.
  • NRF2 expression can be induced by chemotherapeutic agents, hydrogen peroxide, UV and radiation as well as chemicals like brusatol which stimulates NRF2 or ML385 which inhibits NRF2.
  • chemotherapeutic agents hydrogen peroxide, UV and radiation
  • chemicals like brusatol which stimulates NRF2 or ML385 which inhibits NRF2.
  • Example 27 (Prophetic) HOXA5-driven expression of SpCas9 by HOXA5- binding site (HBS) dependent transcription
  • HBS HOXA5- binding site
  • HOXA5 has been identified to influence transcriptional regulation of the pleiotrophin (PTN) gene through binding within the promoter region of the gene (Chen et al., 2005) through HOXA5 core binding motifs (TAAT). This transcriptional activity can be exploited.
  • PTN pleiotrophin
  • TAAT HOXA5 core binding motifs
  • HOXA5 has been shown to be upregulated in EGFR-mutant glioblastoma (Sharanek et al., 2021), therefore, constitutive activity of HOXA5 will initiate PTN promoter-driven transcription of the Cas9 transgene and allow for the ribonucleoprotein complex formation with HOXA5-targeting sgRNAs.
  • Targeting HOXA5 by CRISPR/Cas9 will decrease expression of HOXA5 which will in turn decrease Cas9 expression.
  • Example 28 (Prophetic) Concept of Completekill with dead Cas9 for transcriptional regulation
  • dCas9 inactivated Cas9
  • Dead Cas9 can be targeted to promoters of target genes and act as an activator or repressor of transcription.
  • ARE-driven expression of dCas9 in high NRF2-expressing cells will drive expression of dCas9 targeting NRF2.
  • NRF2 97 ME147020341v.2 130949-01420 expression will be inactivated by dCas9 therefore, reducing expression of NRF2 and reducing expression of Cas9.
  • This offers a temporary solution of reducing oncogenic expression in a gene-dependent manner to allow for coupling with standard of care treatment.
  • SEQ ID NO:2 (tttgattgac atactttgga ggg) [0229] SEQ ID NO:3 (atactttgga ggcaagatat agg) [0230] SEQ ID NO:4 (aggcaagata tagatcttgg agg) [0231] SEQ ID NO:5 (gatatagatc ttggagtaag tgg) [0232] SEQ ID NO:6 (ttcatctagt tgtaactgag cgg) [0233] SEQ ID NO:7 (attcacctgt ctcttcatct agg) [0234] SEQ ID NO:8 (ctcagttaca actagatgaa ggg) [0235] SEQ ID NO:9 (tgaattggga gaaattcacc tgg) [0235] SEQ ID NO:9 (tgaattggga gaaattcacc

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des méthodes et des compositions pour le traitement du cancer à l'aide d'un polynucléotide codant pour une endonucléase Cas d'un système CRISPR/Cas, lié de manière fonctionnelle à une région régulatrice qui est activée par un second polynucléotide ciblé par le système CRISPR/Cas, l'expression du système CRISPR/Cas réduisant l'expression du second polynucléotide.
PCT/US2023/084338 2022-12-16 2023-12-15 Compositions et méthodes pour traiter une grande chimiorésistance par l'intermédiaire de composants régulateurs spécifiques de la chimiorésistance Ceased WO2024130151A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263433237P 2022-12-16 2022-12-16
US63/433,237 2022-12-16

Publications (1)

Publication Number Publication Date
WO2024130151A1 true WO2024130151A1 (fr) 2024-06-20

Family

ID=89772118

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/084338 Ceased WO2024130151A1 (fr) 2022-12-16 2023-12-15 Compositions et méthodes pour traiter une grande chimiorésistance par l'intermédiaire de composants régulateurs spécifiques de la chimiorésistance

Country Status (1)

Country Link
WO (1) WO2024130151A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9925248B2 (en) 2013-08-29 2018-03-27 Temple University Of The Commonwealth System Of Higher Education Methods and compositions for RNA-guided treatment of HIV infection
WO2018232265A1 (fr) * 2017-06-16 2018-12-20 Lonza Ltd Plate-forme de lignée cellulaire mammalienne à autorégulation universelle pour la production de produits biologiques
US20190359973A1 (en) 2017-01-10 2019-11-28 Christiana Care Health Services, Inc. Methods for in vitro site-directed mutagenesis using gene editing technologies
WO2020237208A1 (fr) * 2019-05-23 2020-11-26 Christiana Care Health Services, Inc. Variant nrf2 de gène knockout pour le traitement du cancer
WO2020237186A1 (fr) * 2019-05-23 2020-11-26 Christiana Care Health Services, Inc. Inactivation génique de nrf2 pour le traitement du cancer
US20220017882A1 (en) 2018-12-12 2022-01-20 The Regents Of The University Of California Compositions and methods for modifying a target nucleic acid

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9925248B2 (en) 2013-08-29 2018-03-27 Temple University Of The Commonwealth System Of Higher Education Methods and compositions for RNA-guided treatment of HIV infection
US20190359973A1 (en) 2017-01-10 2019-11-28 Christiana Care Health Services, Inc. Methods for in vitro site-directed mutagenesis using gene editing technologies
WO2018232265A1 (fr) * 2017-06-16 2018-12-20 Lonza Ltd Plate-forme de lignée cellulaire mammalienne à autorégulation universelle pour la production de produits biologiques
US20220017882A1 (en) 2018-12-12 2022-01-20 The Regents Of The University Of California Compositions and methods for modifying a target nucleic acid
WO2020237208A1 (fr) * 2019-05-23 2020-11-26 Christiana Care Health Services, Inc. Variant nrf2 de gène knockout pour le traitement du cancer
WO2020237186A1 (fr) * 2019-05-23 2020-11-26 Christiana Care Health Services, Inc. Inactivation génique de nrf2 pour le traitement du cancer
US20200370041A1 (en) 2019-05-23 2020-11-26 Christiana Care Health Services, Inc. Gene knockout of variant nrf2 for treatment of cancer

Non-Patent Citations (56)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. Gl: 1242863789
AHMAD F ET AL: "Nrf2-driven TERT regulates pentose phosphate pathway in glioblastoma", CELL DEATH & DISEASE, vol. 7, no. 5, 5 May 2016 (2016-05-05), GB, pages e2213 - e2213, XP093150077, ISSN: 2041-4889, DOI: 10.1038/cddis.2016.117 *
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
BALON ET AL., INT. J. MOL. SCI., vol. 23, 2022, pages 573
BATRAKOVA ET AL., J. CONTROL. RELEASE, vol. 219, 2015, pages 396 - 405
BOLD ET AL., SURG. ONCOL., vol. 6, 1997, pages 133 - 42
BOSHART ET AL., CELL, vol. 41, 1985, pages 521 - 30
CHABNER ET AL., NAT. CANCER REV., vol. 5, 2005, pages 65 - 72
CHEN ET AL., BIOTECHNIQUES, vol. 34, 2003, pages 167 - 71
CHEN, H.RUBIN, E.ZHANG, H.CHUNG, S.JIE, C. C.GARRETT, E.BISWAL, S.SUKUMAR, S: "Identification of transcriptional targets of HOXA5", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, no. 19, 2005, pages 19373 - 19380, Retrieved from the Internet <URL:https://doi.org/10.1074/jbc.M413528200>
DAI WEI ET AL: "Cancer gene therapy by NF-[kappa]B-activated cancer cell-specific expression of CRISPR/Cas9 targeting telomeres", GENE THERAPY, NATURE PUBLISHING GROUP, LONDON, GB, vol. 27, no. 6, 7 February 2020 (2020-02-07), pages 266 - 280, XP037177089, ISSN: 0969-7128, [retrieved on 20200207], DOI: 10.1038/S41434-020-0128-X *
DAVIDSON ET AL., NAT. GENET., vol. 3, 1993, pages 219 - 23
DONG ET AL., J. INT. MED. RES., vol. 36, 2008, pages 1273 - 87
FAN JING ET AL: "A Multifunction Lipid-Based CRISPR-Cas13a Genetic Circuit Delivery System for Bladder Cancer Gene Therapy", ACS SYNTHETIC BIOLOGY, vol. 9, no. 2, 21 February 2020 (2020-02-21), Washington DC ,USA, pages 343 - 355, XP093149600, ISSN: 2161-5063, DOI: 10.1021/acssynbio.9b00349 *
FEIGNER ET AL., BETHESDA RES. LAB. FOCUS, vol. 11, no. 2, 1989, pages 25
GELLER ET AL., J. NEUROCHEM., vol. 64, 1995, pages 487 - 96
GELLER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 1149 - 53
GELLER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 7603 - 07
GUERRERO-HUEMELANIASANDRA RAYEGO-MATEOSCRISTINA VAZQUEZ-CARBALLOALEJANDRA PALOMINO-ANTOLINCRISTINA GARCIA-CABALLEROLUCAS OPAZO-RIO: "Protective Role of Nrf2 in Renal Disease.", ANTIOXIDANTS, vol. 10, no. 1, 2020, pages 39 - 10
HAYDEN ET AL., UROL. ONCOL. SEMIN. ORIG. INVESTIG., vol. 32, 2014, pages 806 - 14
HULTQUIST ET AL., CELL REP., vol. 17, 2016, pages 1438 - 52
KAPLITT ET AL., NAT. GENET., vol. 8, 1994, pages 148 - 54
KIM ET AL., J. CONTROL. RELEASE, vol. 266, 2017, pages 8 - 16
KIMMICHAEL P.XINQUN LIDENYING DENGYUN ZHANGBINGBING DAIKENDRA L. ALLTONTARA G. HUGHES ET AL.: "Oncogenic KRAS Recruits an Expansive Transcriptional Network through Mutant P53 to Drive Pancreatic Cancer Metastasis", CANCER DISCOVERY, vol. 11, no. 8, 2021, pages 2094 - 2111
KOONIN ET AL., CURR. OPIN. MICROBIOL., vol. 37, 2017, pages 67 - 78
LE GAL LASALLE ET AL., SCIENCE, vol. 259, 1993, pages 988 - 90
LI ET AL., CANCER GENE THER., vol. 20, 2013, pages 251 - 59
LIN ET AL., ELIFE, vol. 3, 2014, pages 04766
LIU ET AL., J. BIOL. CHEM., vol. 295, 2020, pages 14473 - 87
LUJIAMIAOJAMES A. WILLIAMSJEREMY LUKEFEIJIE ZHANGKIRK CHUMARK A. KAY, A 5' NONCODING EXON CONTAINING ENGINEERED INTRON ENHANCES TRANSGENE EXPRESSION FROM RECOMBINANT AAV VECTORS IN VIVO, 2017
MAKAROVA ET AL., NAT. REV. MICROBIOL., vol. 18, 2020, pages 67 - 83
MANNINO ET AL., BIOTECHNIQUES, vol. 6, 1988, pages 682 - 90
MISHRA RITU ET AL: "Role of NRF2 in immune modulator expression in developing lung", THE FASEB JOURNAL, vol. 35, no. 8, 10 July 2021 (2021-07-10), US, XP093150060, ISSN: 0892-6638, DOI: 10.1096/fj.202100129RR *
NOUNAMO ET AL., MOL. THER. ONCOLYTICS, vol. 6, 2017, pages 90 - 99
QI, L. S.LARSON, M. H.GILBERT, L. A.DOUDNA, J. AWEISSMAN, J. S.ARKIN, A. P.LIM, W. A.: "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression", CELL, vol. 152, no. 5, 2013, pages 1173, XP055346792, Retrieved from the Internet <URL:https://doi.org/10.1016/J.CELL.2013.02.022> DOI: 10.1016/j.cell.2013.02.022
QUANTIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 2581 - 84
ROSENFELD ET AL., CELL, vol. 68, 1992, pages 143 - 55
SAMBROOK J.FRITSCH. E. F.MANIATIS, T: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SANSBURY ET AL., CRISPR J., vol. 2, no. 2, 2019, pages 121 - 32
SCHUMANN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 112, 2015, pages 10437 - 42
SHARANEK, A.BURBAN, A.HERNANDEZ-CORCHADO, A.MADRIGAL, AFATAKDAWALA, I.NAJAFABADI, H. S.SOLEIMANI, V. D.JAHANI-ASL, A.: "Transcriptional control of brain tumor stem cells by a carbohydrate binding protein", CELL REPORTS, vol. 36, no. 9, 2021, pages 109647
SIMPSON ET AL., ONCOLYTIC VIROTHER., vol. 5, 2016, pages 1 - 13
SINGH ET AL., ANTIOXID. REDOX SIGNAL., vol. 13, 2010, pages 1627 - 37
SMITH ET AL., ADV. APPL. MATH, vol. 2, 1981, pages 482 - 89
STRATFORD-PERRICADET ET AL., J. CLIN. INVEST., vol. 90, 1992, pages 626 - 30
STRICH ET AL., J. CLIN. MICROBIOL., vol. 57, 2019, pages 1307 - 18
SUSCHAKJOHN J.LESLEY C. DUPUYCHARLES J. SHOEMAKERCAROLYN SIXSTEVEN A. KWILASKRISTIN W. SPIKJAMES A. WILLIAMSCONNIE S. SCHMALJOHN: "Nanoplasmid Vectors Co-Expressing Innate Immune Agonists Enhance DNA Vaccines for Venezuelan Equine Encephalitis Virus and Ebola Virus", MOLECULAR THERAPY. METHODS & CLINICAL DEVELOPMENT, vol. 17, 2020, pages 810
TAOSHASHASHUE WANGSEYED JAVAD MOGHADDAMAIKSENG OOIELI CHAPMANPAK K WONGDONNA D ZHANG: "Oncogenic KRAS Confers Chemoresistance by Upregulating NRF2", CANCER RESEARCH, vol. 74, no. 24, 2014, pages 7430 - 41, XP055539669, DOI: 10.1158/0008-5472.CAN-14-1439
TORRENTE ET AL., ONCOGENE, vol. 36, 2017, pages 6204 - 12
VERMEIREGILESELIEN DE SMIDTNICK GEUKENSJAMES A. WILLIAMSPAUL DECLERCKKEVIN HOLLEVOET., IMPROVED POTENCY AND SAFETY OF DNA-ENCODED ANTIBODY THERAPEUTICS THROUGH PLASMID BACKBONE AND EXPRESSION CASSETTE ENGINEERING, vol. 32, no. 19-20, 2021, pages 1200 - 1209, Retrieved from the Internet <URL:Https://Home.Liebertpub.Com/Hum>
WANG ET AL., J. CELL. MOL. MED., vol. 24, 2020, pages 3256 - 70
WENNIER ET AL., CURR. PHARM. BIOTECHNOL., vol. 13, 2012, pages 1817 - 33
YANG ET AL., J. VIROL., vol. 69, 1995, pages 2004 - 15
ZACCAGNINI GERMANA ET AL: "Telomerase Mediates Vascular Endothelial Growth Factor-dependent Responsiveness in a Rat Model of Hind Limb Ischemia", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, no. 15, 15 April 2005 (2005-04-15), US, pages 14790 - 14798, XP093149633, ISSN: 0021-9258, DOI: 10.1074/jbc.M414644200 *
ZHANG ET AL., GENOME RES., vol. 7, 1997, pages 649 - 56
ZHOU ET AL., CANCER GENE THER., vol. 23, 2016, pages 1 - 6

Similar Documents

Publication Publication Date Title
US12171813B2 (en) Methods of and compositions for reducing gene expression and/or activity
JP2024041081A (ja) アデノシン塩基編集因子の使用
WO2023215711A9 (fr) Compositions et procédés pour la régulation épigénétique de l&#39;expression de pcsk9
TW202309034A (zh) Dna依賴性蛋白質激酶抑制劑以及其組合物及用途
CA3141313A1 (fr) Variant nrf2 de gene knockout pour le traitement du cancer
US20240279623A1 (en) Compositions and Methods for Making Epigenetic Modifications
US20250381213A1 (en) Methods for lipid nanoparticle delivery of crispr/cas system
US20240209399A1 (en) Systems, methods, and components for rna-guided effector recruitment
JP2024540390A (ja) 可動因子及びそのキメラ構築物
WO2024130151A1 (fr) Compositions et méthodes pour traiter une grande chimiorésistance par l&#39;intermédiaire de composants régulateurs spécifiques de la chimiorésistance
US20250235555A1 (en) Methods for intratumoral delivery of crispr/cas systems
US20250319206A1 (en) Crispr/cas gene editing of neh4 and/or neh5 domains in nrf2
WO2022020192A1 (fr) Compositions et méthodes de ciblage de facteurs de transcription associés à une tumeur
US20240425850A1 (en) Noncanonical crRNA for Highly Efficient Genome Editing
US20250001010A1 (en) Nras gene knockout for treatment of cancer
WO2024182440A2 (fr) Procédés et compositions pour la modification de séquences nucléotidiques
WO2024145615A2 (fr) Compositions et méthodes de régulation épigénétique de l&#39;expression de angptl3
WO2025101979A1 (fr) Compositions et méthodes de régulation épigénétique de gènes pour le traitement de la stéatohépatite non alcoolique ou de la stéatohépatite associée à un dysfonctionnement métabolique
AU2024309386A1 (en) Nras gene knockout for treatment of cancer
WO2025030130A1 (fr) Procédés et compositions comprenant des anticorps se liant à dnmt3a
JP2025525389A (ja) Ｃｉｉｔａ発現のエピジェネティック調節のための組成物及び方法
WO2025264819A1 (fr) Compositions et procédés pour la régulation épigénétique de l&#39;expression de znf410
TW201932479A (zh) 用於ttr基因編輯及治療attr澱粉樣變性之組合物及方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23848163

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23848163

Country of ref document: EP

Kind code of ref document: A1