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WO2024187174A2 - Compositions de traitement du cancer à mutations de kras et leurs utilisations - Google Patents

Compositions de traitement du cancer à mutations de kras et leurs utilisations Download PDF

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Publication number
WO2024187174A2
WO2024187174A2 PCT/US2024/019288 US2024019288W WO2024187174A2 WO 2024187174 A2 WO2024187174 A2 WO 2024187174A2 US 2024019288 W US2024019288 W US 2024019288W WO 2024187174 A2 WO2024187174 A2 WO 2024187174A2
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kras
individual
cell
seq
cancer
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WO2024187174A3 (fr
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Gilles Divita
Neil P. Desai
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Aadigen LLC
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Aadigen LLC
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Priority to PCT/US2025/019236 priority Critical patent/WO2025193628A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • the present application relates to guide RNAs and genome-editing complexes or nanoparticles that are useful for specifically targeting a mutated KRAS.
  • KRAS-G12C inhibitors sotorasib and adagrasib are used to treat patients with advanced nonsmall cell lung cancer (NSCLC) carrying KRAS-G12C mutations.
  • NSCLC nonsmall cell lung cancer
  • KRAS-G12C inhibitor therapy mainly due to intrinsic or acquired resistance caused by cellular, molecular, and genetic mechanisms. See e.g., Cancer Gene Ther 29, 875-878 (2022).
  • the present application in one aspect provides a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a- 1) a cellpenetrating peptide, and a-2) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, and 273-341, optionally wherein the complex comprises a single KRAS guide RNA, further optionally wherein the complex comprises a single guide RNA targeting a single mutation; or b-1) a cell-penetrating peptide, and b-2) a RNAi targeting a mutated KRAS, optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229 and 231-234, further optionally wherein the RNAi comprises
  • the individual comprises a secondary mutation in KRAS, optionally wherein the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation; 2) the cancer comprises a copy number variation in KRAS; 3) the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy; 4) the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter; and/or 5) the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP- bound wildtype RAS, optionally where
  • the present application in another aspect provides a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a- 1) a cellpenetrating peptide, and a-2) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, and 273-341, optionally wherein the complex comprises a single KRAS guide RNA, further optionally wherein the complex comprises a single guide RNA targeting a single mutation; or b-1) a cell-penetrating peptide, and b-2) a RNAi targeting a mutated KRAS, optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229 and 231-234, further optionally wherein the RNAi comprises
  • the complex comprises a single guide RNA targeting a single mutation in KRAS.
  • the cellpenetrating peptide is selected from the group consisting of an ADGN-100 peptide and an ADGN-106 peptide (i.e., VEPEP-6 peptide), optionally wherein: the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135- 175, 259-260, 267-269; and/or the ADGN-106 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-107, 111-117, 261-266, 270, 272 and 353-355.
  • the cancer is resistant, refractory or recurrent to the KRAS inhibitor, further optionally the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the patient who has been treated, showed a response and then eventually progressed as the cancer no longer responded to the KRAS inhibitor is deemed as an example of having a cancer that is recurrent to the KRAS inhibitor.
  • the R68M, Y96D, or A59T mutation is a somatic mutation, and optionally wherein the individual developed the R68M, Y96D, or A59T mutation after the KRAS inhibitor treatment, optionally wherein the method further comprises determining whether the individual has the R68M, Y96D, or A59T mutation and/or selecting the individual for treatment due to the presence of the R68M, Y96D, or A59T mutation.
  • the KRAS inhibitor specifically binds to the mutant KRAS protein
  • the KRAS inhibitor is selected from the group consisting of MRTX1133, RMC-9805, sotorasib, adagrasib, ganetespib, RMC-6236, YL- 17231, BDTX-4933, QTX3034, ABT-200, ADT-1004, AN9025, OC211, JAB-23425, BI-2865, BI-2493, ABREV01, A2A-03, LY3537982, and LY-4066434, optionally wherein the KRAS inhibitor is selected from the group consisting of MRTX1133, RMC-9805, sotorasib, adagrasib, and ganetespib.
  • the complex further comprises a second cell penetrating peptide (CPP), optionally wherein the second CPP is a ADGN-100 or ADGN-106 peptide, optionally wherein: the complex comprises 1) a first cell-penetrating peptide, 2) a second cell-penetrating peptide, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA), wherein the individual has been subjected to a KRAS inhibitor, and further optionally wherein the first cell-penetrating peptide comprises the amino acid sequence of any one of SEQ ID NOs: 90 and 154, and further optionally wherein the second cell-penetrating peptide comprises the amino acid sequence of any one of SEQ ID NOs: 96, 162, 353, and 354.
  • CPP cell penetrating peptide
  • the complex comprises 1) a first cell-penetrating peptide
  • the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA), b) the nucleotide sequence substantially complementary to a target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34, c) the guide RNA has a length of no more than about 200, 100, 50, 40, 30, 28, or 25 nucleotides, and/or d) the nucleotide is chemically modified.
  • auxiliary trans-activating crRNA auxiliary trans-activating crRNA
  • the nucleotide sequence is 100% complementary to the target sequence, optionally wherein the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34, optionally wherein nucleotide sequence has the same length as the target sequence.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease, wherein optionally the DNA nuclease is selected from the group consisting of a CRISPR- associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof, optionally wherein the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • Cas CRISPR- associated protein
  • ZFN zinc finger nuclease
  • TALEN transcription activatorlike effector nuclease
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the cellpenetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP- 9 peptides, and ADGN-100 peptides.
  • the cellpenetrating peptide further comprises one or more moieties covalently linked to N-terminus of the cell-penetrating peptide, and wherein the one or more moieties are selected from the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, a linker moiety, and a targeting moiety, optionally wherein: a) the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide, and/or b) the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the cell-penetrating peptide, optionally wherein the targeting peptide is selected from the group consisting of SEQ
  • the cellpenetrating peptide comprises, from N-terminus, an acetyl group, a targeting moiety and a linker moiety covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cellpenetrating peptide further comprises a carbohydrate moiety, optionally wherein the carbohydrate moiety is GalNAc.
  • the cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-107, 111-117, 153-175, 272, 353-355, optionally wherein the cellpenetrating peptide comprising SEQ ID NO: 162, 272, and 353-355.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1
  • the molar ratio of the cell -penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1
  • the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • the method further comprises one or more additional guide RNAs comprising different guide sequences, optionally wherein at least two of the two or more guide RNAs target one single KRAS mutation, further optionally wherein at least two of the two or more guide RNAs target two or more different KRAS mutations, further optionally wherein at least two of the two or more guide RNAs target G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, and/or A146T.
  • the average diameter of the complex is between about 10 nm and about 300 nm.
  • the complex is in a nanoparticle.
  • the method comprises administering two or more complexes, wherein the two or more complexes comprise different guide RNAs that target different KRAS mutations.
  • the individual comprises a G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, and/or A146T mutation.
  • the individual does not develop a secondary KRAS mutation in any of the exons after the KRAS treatment.
  • the present application in another aspect provides a non-naturally occurring polynucleotide comprising a guide RNA for targeting mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 273-341.
  • the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • the polynucleotide is chemically modified.
  • the present application in another aspect provides a genome-editing complex comprising a) a first cell-penetrating peptide, and b) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprises a polynucleotide as described herein.
  • the genome-editing complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease is selected from the group consisting of a CRISPR-associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof.
  • the DNA nuclease comprises a Cas polypeptide, optionally wherein the Cas polypeptide is Cas9.
  • the genome-editing complex further comprises a second cell penetrating peptide that is distinct from the first cell penetrating peptide, optionally the second cell penetrating peptide is selected from VEPEP-6 peptides and ADGN-100 peptides.
  • the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the first cell-penetrating peptide further comprises one or more moieties covalently linked to N-terminus of the first cell-penetrating peptide, and wherein the one or more moieties are selected from the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, a linker moiety, and a targeting moiety.
  • the first cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide.
  • the first cellpenetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide.
  • the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the first cellpenetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the first cell-penetrating peptide comprises, from N-terminus, an acetyl group, a targeting moiety and a linker moiety covalently linked to the N-terminus of the first cell-penetrating peptide.
  • the first cell-penetrating peptide further comprises a carbohydrate moiety.
  • the carbohydrate moiety is GalNAc.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-107, 111-117, 135-175, 259-270, 272, and 353-355.
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1, optionally wherein the molar ratio of the first cellpenetrating peptide to the guide RNA is between about 1:1 and about 20:1. In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1. In some embodiments, the genome-editing complex further comprises one or more additional guide RNAs comprising different guide sequences. In some embodiments, at least two of the two or more guide RNAs target one single KRAS mutation.
  • At least two of the two or more guide RNAs target two or more different KRAS mutations. In some embodiments, at least two of the two or more guide RNAs target G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, A18D, KI 17N, and/or A146T. In some embodiments, the average diameter of the genome-editing complex is between about 10 nm and about 300 nm.
  • the present application in another aspect provides a nanoparticle comprising a core comprising the genome-editing complex described herein.
  • the present application in another aspect provides a pharmaceutical composition
  • a pharmaceutical composition comprising the guide RNA described herein, the genome-editing complex described herein, or the nanoparticle described herein and a pharmaceutically acceptable carrier, optionally wherein the composition comprises two or more nanoparticles, wherein the two or more nanoparticles comprise different guide RNAs that target different KRAS mutations.
  • the present application in another aspect provides a method of preparing the genomeediting complex described herein, comprising combining the first cell-penetrating peptide with the guide RNA, thereby forming the genome-editing complex.
  • the present application in another aspect provides a method of modifying mutated KRAS in a cell, comprising contacting the cell with guide RNA described herein, the genomeediting complex described herein, or the nanoparticle described herein.
  • the present application in another aspect provides a method of treating an individual a cancer in an individual comprising administering to the individual a complex comprising the genome-editing complex described herein, the nanoparticle described herein, or the pharmaceutical composition described herein.
  • FIGs. 1A-1B show proliferation rates of H358 cells and five stable sotorasib resistant H358 clones under the treatment of sotorasib or adagrasib at different concentrations.
  • the five stable sotorasib resistant H358 clones are H-358-C1, H-358-C2, H358-C3 (KRAS G12C/Y96D), H-358-C4 (KRAS G12C/R68M) and H-358-C5 (KRAS G12C/A59T).
  • H358-C3 KRAS G12C/Y96D
  • H-358-C4 H-358-C4
  • H-358-C5 KRAS G12C/A59T
  • FIG. 2A shows proliferation rates of H358 cells and five stable sotorasib resistant H358 clones under the treatment of an exemplary complex ADGN-122 that has a gRNA specifically targets G12C complexed with a cell-penetrating peptide.
  • FIG. 2B shows a western blot result of the level of p-ERK in the H-358 and H-358 resistant clones cells after treatment of ADGN-122, sororasib or adagrasib.
  • FIG. 2C shows relative levels of KRAS G12C gene expression in parental and AMG 510-resistant H-358 clones.
  • FIG. 2D shows the ratio of KRAS G12C -GTP in parental and AMG 510-resistant H-358 clones. The ratio is calculated as KRAS G12C over RAS over GAPDH.
  • FIG. 3 shows proliferation rates of H358 cells and three resistant H358 clones under the treatment of an exemplary complex that has siRNA specifically targeting G12C complexed with a cell-penetrating peptide.
  • FIG. 4 depicts a summary of IC50 of different treatments against H358 cells and H358 resistant clones.
  • FIG. 5A shows relative levels of KRAS gene expression in parental and MTRX-1133- resistant PANC-1 and ASPC-1 cells.
  • FIG. 5B shows protein expression levels of KRAS G12D , relative to GAPDH protein expression levels, in parental and MTRX-1133-resistant PANC-1 and ASPC-1 cells.
  • FIG. 5C shows the ratio of KRAS G12D -GTP in parental and MTRX-1133- resistant PANC-1 and ASPC-1 cells as determined by a pulldown assay. The ratio is calculated as KRAS G12D over RAS over GAPDH.
  • FIG. 6A shows proliferation rates of parental and MTRX-1133-resistant PANC-1 and ASPC-1 cells under the treatment of an ADGN-123 nanoparticle that has a gRNA specifically targeting KRAS G12D associated with an ADGN peptide.
  • FIG. 6B shows proliferation rates of parental and MTRX-1133-resistant PANC-1 and ASPC-1 cells under the treatment of MTRX-1133.
  • FIG. 6C is a table of the IC50 of cell lines treated with either ADGN-121 or MTRX-1133. Fines labeled as PANC-1 and ASPC-1 are the parental cell lines. Cell lines denoted with Cl, C2, or C3 are the MTRX-1133-resistant cell lines.
  • FIG. 7 shows the expression efficiency of ADGN/Cas9mRNA/sgRNA complexes in PANC-1 cells.
  • the complexes contained only ADGN-100, only ADGN-106, ADGN-100 mixed with the indicated targeting peptide (ADGN-100-hydro-3, ADGN-106-hydro-3, ADGN-1088, or ADGN-108-R91), or ADGN-106 mixed with the indicated targeting peptide (ADGN-100-hydro-3, ADGN-106-hydro-3, ADGN-1088, or ADGN-108-R91).
  • the complexes were mixed with either 0.1 pg or 0.5 pg of CAS9 mRNA.
  • FIG. 8 shows target sequences for the design of sgRNA targeting KRAS with a G12D, G12V, G12C, G12R, G12S, G12A, G13D, G13C, Q61H, Q61L, A18D, KI 17N, or A146T mutation.
  • the present application in one aspect provides methods of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cellpenetrating peptide, and b) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1- 37, 241-257, 271, and 273-341; wherein the individual has been subjected to a KRAS inhibitor treatment.
  • crRNA specificitydetermining CRISPR RNA
  • the present application in another aspect provides methods of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide, and b) a RNAi targeting a mutated KRAS, optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229 and 231-234, further optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual has been subjected to a KRAS inhibitor treatment.
  • the present application in another aspect provides methods of treating a cancer in an individual comprising administering to the individual a complex comprising: a- 1) a cellpenetrating peptide, and a-2) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, and 273-341; or b-1) a cell-penetrating peptide, and b-2) a RNAi targeting a mutated KRAS, optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229 and 231-234, further optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229, wherein: 1) the individual comprises a R68, Y
  • the present application in another aspect provides guide RNAs and complexes and nanoparticles comprising same.
  • the present application provides a non-naturally occurring polynucleotide comprising a guide RNA for targeting mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 273-341.
  • crRNA specificity-determining CRISPR RNA
  • the present application is at least partly based upon the advantageous effects demonstrated by the methods described herein that involve using a complex comprising a guide RNA or siRNA specifically targeting a mutant KRAS complexed with a cell-penetrating peptide as described herein.
  • a complex comprising a guide RNA or siRNA specifically targeting a mutant KRAS complexed with a cell-penetrating peptide as described herein.
  • such exemplary complexes effectively inhibited the proliferation of cancer cells harboring KRAS mutation that are resistant to recently approved drugs sotorasib and/or atagrasib as well as other inhibitors (e.g., MRTR-1133).
  • the exemplary complexes that involve a guide RNA that targets a specific mutation in KRAS were able to inhibit proliferation of resistant clones that involve a secondary mutation (e.g., H358-G12C/Y96D, H358- G12C/R68M, H358-G12C/A59T) and resistant clones that do not involve a secondary mutation (e.g., H358-C1 and H358-C2).
  • the exemplary complexes inhibit proliferation of inhibitor-resistant clones characterized by upregulated KRAS transcription levels (KRAS mRNA) and/or increased activation of the wildtype RAS pathway, of which the activity is independent of mutant KRAS. See, e.g., Example 4, FIGs. 2C and 2D; Example 7, FIGs. 5B and 5C.
  • KRAS mRNA upregulated KRAS transcription levels
  • compositions and methods discussed in the present application serve as promising treatments for patients a) who are resistant to a KRAS inhibitor, such as sotorasib, adagrasib, MRTX1133, RMC-9805, or ganetespib, b) who harbor an additional KRAS aberration other than the specific mutation targeted by the specific guide RNA in the composition, and c) who have an abnormal RAS pathway (e.g., overexpressed H-RAS and/or N-RAS protein).
  • WO 2021/217100 and WO 2019/079215 are incorporated herein for all their entirety.
  • the term “guide RNA” refers to a polynucleotide that cleaves, inserts, or links a target DNA in a cell via RNA editing.
  • the guide RNA may be a single-chain guide RNA (sgRNA).
  • the guide RNA may be a CRISPR RNA (crRNA) specific to the target nucleotide sequence.
  • the guide RNA may further include a trans-activating crRNA (tracrRNA) interacting with Cas9 nuclease.
  • the tracrRNA may include a polynucleotide forming a loop structure.
  • the guide RNA may have a length of 10 nucleotides to 30 nucleotides.
  • the guide RNA may have a length of, for example, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides.
  • the guide RNA may include RNA, DNA, PNA, or a combination thereof.
  • the guide RNA may be chemically modified.
  • the guide RNA may be a component of molecular scissors (programmable nuclease).
  • the molecular scissor refers to all types of nucleases capable of recognizing and cleaving a specific site on the genome.
  • the molecular scissors may be, for example, transcription activator-like effector nuclease (TALEN), zinc-finger nuclease, meganuclease, RNA-guided engineered nuclease (RGEN), Cpfl, and Ago homolog (DNA-guided endonuclease).
  • the RGEN refers to a nuclease including a guide RNA specific to a target DNA and Gas protein as components.
  • the polynucleotide may be, for example, a component of RGEN.
  • single guide RNA or “sgRNA” refers to a polynucleotide sequence comprising a guide sequence, a tracr sequence and a tracr mate sequence.
  • guide sequence refers to the about 20 bp sequence within the guide RNA that specifies the target site.
  • tracr mate sequence may also be used interchangeably with the term “direct repeat(s)”.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
  • Polynucleotide refers to polymers of nucleotides of any length, and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA and RNA.
  • DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors (PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups.
  • RNA may be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, RNA, viral RNA (vRNA), and combinations thereof.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including for example locked nucleic acid (LNA), unlocked nucleic acid (UNA), and zip nucleic acid (ZNA), which can be synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
  • LNA locked nucleic acid
  • UNA unlocked nucleic acid
  • ZNA zip nucleic acid
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer e al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al, J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).
  • "Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylases, and alkylhalides.
  • Oligonucleotide generally refers to short, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • CRISPR system refers collectively to proteins, transcripts and other molecules involved in the activity of CRISPR-associated (“Cas”) nucleases (such as RNA- guided endonucleases, or “RGENs”), including Cas gene products, Cas gene sequences, tracr (trans-activating CRISPR) sequences (e.g.
  • tracrRNA or an active partial tracrRNA include 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, transcripts, and products derived from a CRISPR locus.
  • one or more molecules of a CRISPR system are derived from a type I, type II, or type III CRISPR system.
  • one or more molecules of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a CRISPR system is characterized by molecules 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 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. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is present in the nucleus or cytoplasm of a cell.
  • the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.
  • a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template,” “editing polynucleotide,” “editing sequence,” “donor sequence,” or “donor nucleic acid”.
  • an exogenous template polynucleotide may be referred to as an editing template.
  • the recombination is homologous recombination.
  • 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, 20, 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.
  • a wild-type tracr sequence may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.
  • 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.
  • the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.
  • one or more molecules of a CRISPR system are introduced into a host cell such that formation of a CRISPR complex at one or more target sites can occur.
  • a Cas nuclease, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be introduced into a host cell to allow formation of a CRISPR complex at a target sequence in the host cell complementary to the guide sequence.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%/, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993). Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”. Elsevier, N.Y.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.
  • expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • the terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • the terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject.
  • the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
  • treatment refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • the term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein.
  • the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
  • Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
  • compositions and methods of the present application may comprise, consist of, or consist essentially of the essential elements and limitations of the application described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful.
  • the present application in one aspect provides a method of treating a disease (such as a cancer) in an individual comprising administering to the individual a complex or nanoparticle comprising a complex as described herein.
  • a disease such as a cancer
  • the present application in another aspect provides a method of modifying mutated KRAS in a cell comprising contacting the cell with the complex or nanoparticle comprising a complex as described herein.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29- 37 and 273 (e.g., SEQ ID NO: 29, 31, 33, 34, e.g., SEQ ID NO: 29), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12C inhibitor (e.g., sotorasib or aragrasib).
  • CCPP cell-penetrating peptide
  • crRNA specificitydetermining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29-37 and 273 (e.g., SEQ ID NO: 29, 31, 33, 34, e.g., SEQ ID NO: 29), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12D inhibitor (e.g., MRTX1133 or RMC-9805).
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29-37 and 273 (e.g., SEQ ID NO: 29, 31, 33, 34, e.g., SEQ ID NO: 29), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12V inhibitor (e.g., ganetespib).
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175, 259-260, and 267-269 (e.g., SEQ ID NOs: 157-167, 169-171, 259-260 and 267-269, e.g., SEQ ID NO: 162).
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89- 90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cancer is resistant, refractory or recurrent to the KRAS inhibitor.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a R68, Y96, or A59 mutation, optionally the individual comprises a R68M, Y96D, or A59T mutation on KRAS.
  • the method further comprises determining whether the individual has the R68M, Y96D, or A59T mutation and/or selecting the individual for treatment due to the presence of the R68M, Y96D, or A59T mutation.
  • the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment. In some embodiments, the complex is administered no more about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment. In some embodiments, the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9. In some embodiments, the complex is in a nanoparticle. In some embodiments, individual does not develop a secondary KRAS mutation in any of the exons after the KRAS inhibitor treatment.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cellpenetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15- 28 (e.g., SEQ ID NO: 15, 16, 19-21, 23, e.g., SEQ ID NO: 19), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12C inhibitor (e.g., sotorasib or aragrasib).
  • CCPP cell-penetrating peptide
  • crRNA specificitydetermining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15-28 (e.g., SEQ ID NO: 15, 16, 19-21, 23, e.g., SEQ ID NO: 19), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12D inhibitor (e.g., MRTX1133 or RMC-9805).
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • a method of treating a cancer in an Individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15-28 (e.g., SEQ ID NO: 15, 16, 19-21, 23, e.g., SEQ ID NO: 19), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12V inhibitor (e.g., ganetespib).
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272 and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: il).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cancer is resistant, refractory or recurrent to the KRAS inhibitor.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a R68, Y96, or A59 mutation, optionally the individual comprises a R68M, Y96D, or A59T mutation on KRAS.
  • the method further comprises determining whether the individual has the R68M, Y96D, or A59T mutation and/or selecting the individual for treatment due to the presence of the R68M, Y96D, or A59T mutation.
  • the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment. In some embodiments, the complex is administered no more about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment. In some embodiments, the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9. In some embodiments, the complex is in a nanoparticle. In some embodiments, individual does not develop a secondary KRAS mutation in any of the exons after the KRAS inhibitor treatment.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N- terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14 (e.g., SEQ ID NO: 1, 3, 4, 6, 8, e.g., SEQ ID NO: 3, 6, or 8, e.g., SEQ ID NO: 3), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12C inhibitor (e.g., sotorasib or aragrasib).
  • crRNA specificitydetermining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14 (e.g., SEQ ID NO: 1, 3, 4, 6, 8, e.g., SEQ ID NO: 3, 6, or 8, e.g., SEQ ID NO: 3), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12D inhibitor (e.g., MRTX1133 or RMC-9805).
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14 (e.g., SEQ ID NO: 1, 3, 4, 6, 8, e.g., SEQ ID NO: 3, 6, or 8, e.g., SEQ ID NO: 3), wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12V inhibitor (e.g., ganetespib).
  • CRISPR RNA specificitydetermining CRISPR RNA
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN- 106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353- 355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cancer is resistant, refractory or recurrent to the KRAS inhibitor.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a R68, Y96, or A59 mutation, optionally the individual comprises a R68M, Y96D, or A59T mutation on KRAS.
  • the method further comprises determining whether the individual has the R68M, Y96D, or A59T mutation and/or selecting the individual for treatment due to the presence of the R68M, Y96D, or A59T mutation.
  • the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment. In some embodiments, the complex is administered no more about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment. In some embodiments, the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9. In some embodiments, the complex is in a nanoparticle. In some embodiments, individual does not develop a secondary KRAS mutation in any of the exons after the KRAS inhibitor treatment.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cellpenetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cellpenetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 29-37, 253-257, 271, and 273 (e.g., SEQ ID NO: 34), wherein the individual comprises a G12C mutation (e.g., in the cancer).
  • CCP cell-penetrating peptide
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, R68, Y96, A59, A18D, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP- bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N- RAS.
  • the CPP further comprises a targeting moiety fused to the N- terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN-106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 15-28, and 247-252 (e.g., SEQ ID NO: 19), wherein the individual comprises a G12D mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, R68, Y96, A59, A18D, K117N, and/or Al 46T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP- bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N- RAS.
  • the CPP further comprises a targeting moiety fused to the N- terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN-106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 1-14 and 241-246 (e.g., SEQ ID NO: 3), wherein the individual comprises a G12V mutation (e.g., in the cancer).
  • CCP cell-penetrating peptide
  • DNA nuclease e.
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, R68, Y96, A59, A18D, K117N, and/or Al 46T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP- bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N- 1 RAS.
  • the CPP further comprises a targeting moiety fused to the N- terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN-106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 284-294, wherein the individual comprises a G12A mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nucle
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12R, G12S, G13D, G13C, Q61H, Q61L, R68, Y96, A59, A18D, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 274-283, wherein the individual comprises a G12R mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nucle
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12S, G13D, G13C, Q61H, Q61L, R68, Y96, A59, A18D, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 295-304, wherein the individual comprises a G12S mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nucle
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12R, G13D, G13C, Q61H, Q61L, R68, Y96, A59, A18D, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 305-309, wherein the individual comprises a G13D mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nucle
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12R, G12S, G13C, Q61H, Q61L, R68, Y96, A59, A18D, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 310-315, wherein the individual comprises a G13C mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nucle
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12R, G12S, G13D, Q61H, Q61L, R68, Y96, A59, A18D, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 316-322, wherein the individual comprises a Q61H mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nuclea
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61L, R68, Y96, A59, A18D, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 323-329, wherein the individual comprises a Q61L mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nucle
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, R68, Y96, A59, A18D, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 330-332, wherein the individual comprises a A18D mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nucle
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, R68, Y96, A59, K117N, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Ann, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, R68, Y96, A59, A18D, and/or A146T mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising an ADGN-100 peptide or an ADGN-106 peptide, 2) a DNA nuclease (e.g., a CRISPR-associated DNA nuclease or an mRNA encoding a CRISPR- associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS, wherein the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (e.g., 100% complementary) to a target sequence selected from the group consisting of SEQ ID Nos: 336-341, wherein the individual comprises a A147T mutation (e.g., in the cancer).
  • CCPP cell-penetrating peptide
  • DNA nuclease e.g., a CRISPR-associated DNA nuclea
  • the individual comprises at least one more KRAS mutation.
  • the individual further comprises one or more of G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, R68, Y96, A59, A18D, and/or K117N mutation.
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor.
  • the guide RNA has the same length as the target sequence.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS.
  • the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the CPP further comprises a targeting moiety fused to the N-terminus of the ADGN-100 or ADGN106 peptide.
  • the targeting moiety is fused to the ADGN-100 or ADGN- 106 peptide via a linker moiety.
  • the ADGN-100 or ADGN-106 peptide comprises an amino acid sequence of 137, 138, 89 or 355.
  • the targeting moiety comprises an amino acid sequence of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., any of SEQ ID NO: 201, 351, or 352, 423-426).
  • the linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3), Aun, Ava, and Ahx.
  • the CPP (the first CPP) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 96, 261, 272, 162, 353, 354, and 419-422.
  • the complex further comprises a second CPP that is distinct from the first CPP.
  • the first CPP comprises a sequence selected from SEQ ID Nos; 96, 261, 272, 162, 353, and 354, and the second CPP comprises an ADGN-100 or ADGN-106 peptide that does not have a targeting moiety.
  • the second CPP comprises a sequence of SEQ ID NO: 137, 138, 89 or 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12C, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12C inhibitor (e.g., sotorasib or aragrasib).
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12C, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12D inhibitor (e.g., MRTX1133 or RMC-9805).
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12C, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12C, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12V inhibitor (e.g., ganetespib).
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175. 259-260, and 267-269 (e.g., SEQ ID NOs: 157-167, 169-171, 259-260 and 267-269, e.g., SEQ ID NO: 162).
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89- 90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135- 175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cancer is resistant, refractory or recurrent to the KRAS inhibitor.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a R68, Y96, or A59 mutation, optionally the individual comprises a R68M, Y96D, or A59T mutation on KRAS.
  • the method further comprises determining whether the individual has the R68M, Y96D, or A59T mutation and/or selecting the individual for treatment due to the presence of the R68M, Y96D, or A59T mutation.
  • the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • the complex is administered no more about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the complex is in a nanoparticle. In some embodiments, individual does not develop a secondary KRAS mutation in any of the exons after the KRAS inhibitor treatment.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N- terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12D, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12C inhibitor (e.g., sotorasib or aragrasib).
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12D, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12D inhibitor (e.g., MRTX1133 or RMC-9805).
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12D
  • the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234
  • the individual has been subjected to a KRAS inhibitor treatment
  • the KRAS inhibitor is a G12D inhibitor (e.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12D, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12V inhibitor (e.g., ganetespib).
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175. 259-260, and 267-269 (e.g., SEQ ID NOs: 157-167, 169-171, 259-260 and 267-269, e.g., SEQ ID NO: 162).
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89- 90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135- 175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cancer is resistant, refractory or recurrent to the KRAS inhibitor.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a R68, Y96, or A59 mutation, optionally the individual comprises a R68M, Y96D, or A59T mutation on KRAS.
  • the method further comprises determining whether the individual has the R68M, Y96D, or A59T mutation and/or selecting the individual for treatment due to the presence of the R68M, Y96D, or A59T mutation.
  • the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • the complex is administered no more about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the complex is in a nanoparticle. In some embodiments, individual does not develop a secondary KRAS mutation in any of the exons after the KRAS inhibitor treatment.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N- terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12V, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12C inhibitor (e.g., sotorasib or aragrasib).
  • CPP cell-penetrating peptide
  • KRAS inhibitor is a G12C inhibitor
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12V, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12D inhibitor (e.g., MRTX1133 or RMC-9805).
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12V
  • the individual has been subjected to a KRAS inhibitor treatment
  • the KRAS inhibitor is a G12D inhibitor (e.g., MRTX1133 or RMC-9805).
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12V, wherein the individual has been subjected to a KRAS inhibitor treatment, wherein the fRAS inhibitor is a G12V inhibitor (e.g., ganetespib).
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12V
  • the individual has been subjected to a KRAS inhibitor treatment
  • the fRAS inhibitor is a G12V inhibitor (e.g., ganetespib).
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63- 107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261- 266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cancer is resistant, refractory or recurrent to the KRAS inhibitor.
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a R68, Y96, or A59 mutation, optionally the individual comprises a R68M, Y96D, or A59T mutation on KRAS.
  • the method further comprises determining whether the individual has the R68M, Y96D, or A59T mutation and/or selecting the individual for treatment due to the presence of the R68M, Y96D, or A59T mutation.
  • the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment. In some embodiments, the complex is administered no more about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment. In some embodiments, the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9. In some embodiments, the complex is in a nanoparticle. In some embodiments, individual does not develop a secondary KRAS mutation in any of the exons after the KRAS inhibitor treatment.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N- terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Ann, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29- 37 and 273 (e.g., SEQ ID NO: 29, 31, 33, 34, e.g., SEQ ID NO: 29), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CCPP cell-penetrating peptide
  • crRNA specificitydetermining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29-37 and 273 (e.g., SEQ ID NO: 29, 31, 33, 34, e.g., SEQ ID NO: 29), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation .
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29-37 and 273 (e.g., SEQ ID NO: 29, 31, 33, 34, e.g., SEQ ID NO: 29), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • the R68, Y96, or A59 mutation is a somatic mutation. In some embodiments, the R68, Y96, or A59 mutation is a germline mutation. In some embodiments, the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9. In some embodiments, the complex is in a nanoparticle.
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN- 100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196- 205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1 : 1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15- 28 (e.g., SEQ ID NO: 15, 16, 19-21, 23, e.g., SEQ ID NO: 19), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CCPP cell-penetrating peptide
  • crRNA specificitydetermining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15-28 (e.g., SEQ ID NO: 15, 16, 19-21, 23, e.g., SEQ ID NO: 19), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15-28 (e.g., SEQ ID NO: 15, 16, 19-21, 23, e.g., SEQ ID NO: 19), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • the R68, Y96, or A59 mutation is a somatic mutation. In some embodiments, the R68, Y96, or A59 mutation is a germline mutation. In some embodiments, the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9. In some embodiments, the complex is in a nanoparticle.
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN- 100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196- 205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1 : 1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14 (e.g., SEQ ID NO: 1, 3, 4, 6, 8, e.g., SEQ ID NO: 3, 6, or 8, e.g., SEQ ID NO: 3), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CCPP cell-penetrating peptide
  • crRNA specificitydetermining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14 (e.g., SEQ ID NO: 1, 3, 4, 6, 8, e.g., SEQ ID NO: 3, 6, or 8, e.g., SEQ ID NO: 3), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14 (e.g., SEQ ID NO: 1, 3, 4, 6, 8, e.g., SEQ ID NO: 3, 6, or 8, e.g., SEQ ID NO: 3), wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation .
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175. 259-260, and 267-269 (e.g., SEQ ID NOs: 157-167, 169-171, 259-260 and 267-269, e.g., SEQ ID NO: 162).
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353- 355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the R68, Y96, or A59 mutation is a somatic mutation.
  • the R68, Y96, or A59 mutation is a germline mutation.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the complex is in a nanoparticle.
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175. 259-260, and 267-269 (e.g., SEQ ID NOs: 157-167, 169-171, 259-260 and 267-269, e.g., SEQ ID NO: 162).
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353- 355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196- 205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1 : 1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12C, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12C, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12C, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation .
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12C, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the R68, Y96, or A59 mutation is a somatic mutation.
  • the R68, Y96, or A59 mutation is a germline mutation.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the complex is in a nanoparticle.
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63- 107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261- 266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N- terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety,
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12D, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12D, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M,
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12D, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12D, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M,
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12D, optionally wherein the siRNA comprises a sequence set forth in any of SEQ ID NOs: 233-234, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the R68, Y96, or A59 mutation is a somatic mutation.
  • the R68, Y96, or A59 mutation is a germline mutation.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the complex is in a nanoparticle.
  • the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63- 107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261- 266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N- terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12V, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12V
  • the individual comprises a R68, Y96, or A59 mutation on KRAS
  • optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12V, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a siRNA targeting G12V
  • the individual comprises a R68, Y96, or A59 mutation on KRAS
  • optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP), and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a siRNA targeting G12V, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation .
  • the CPP is an ADGN- 100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the R68, Y96, or A59 mutation is a somatic mutation.
  • the R68, Y96, or A59 mutation is a germline mutation.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the complex is in a nanoparticle.
  • the cellpenetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP- 9 peptides, and ADGN-100 peptides.
  • the CPP is an ADGN-100 peptide or ADGN-106 peptide.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 135-175.
  • the CPP comprises a sequence set forth in any one of SEQ ID NOs: 63-107, 111-117, 261-268, 270, 272, and 353-355 (e.g., SEQ ID NOs: 89-90, 92-107, 112, 261-266, 270, 272, and 353-355, e.g., SEQ ID NO: 272).
  • the CPP comprises a sequence set for in any one of SEQ ID NOs: 367-377, 387-396, and 418.
  • the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1 : 1 and about 80:1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 162 or 272, and b) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NO: 29, wherein the cancer is resistant to a KRAS inhibitor treatment, wherein the KRAS inhibitor is a G12C inhibitor (e.g., sotorasib or aragrasib).
  • CCPP cell-penetrating peptide
  • crRNA specificity-determining CRISPR RNA
  • the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the complex is in a nanoparticle.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a) a cell-penetrating peptide (CPP) comprising the sequence set forth in SEQ ID NO: 162 or 272, and b) a RNAi targeting a mutated KRAS, wherein the RNAi comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • CPP cell-penetrating peptide
  • a RNAi targeting a mutated KRAS wherein the RNAi comprises a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual comprises a R68, Y96, or A59 mutation on KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • the complex is in a nanoparticle.
  • the cancer is resistant to both sotorasib and adagrasib.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a cell-penetrating peptide (CPP) comprising from N-terminus to C-terminus a) a targeting moiety comprising an amino acid sequence of any one of 196-205, 235-240, 351-352, 356-366, 378-386, 397- 417, and 423-426 (e.g., SEQ ID NO: 201, 351, or 352), and b) an amino acid sequence of any one of SEQ ID Nos: 137, 138, 89 and 355, 2) a DNA nuclease (e.g., a CRIS PR-associated DNA nuclease or an mRNA encoding a CRIS PR-associated DNA nuclease), and 3) a guide RNA targeting a mutated KRAS.
  • CCPP cell-penetrating peptide
  • a targeting moiety comprising an amino acid sequence of any one of 196-205,
  • the individual comprises a G12D, G12V, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, and/or A146T mutation (e.g., in the cancer).
  • the individual has been subjected to a KRAS inhibitor (e.g., KRAS inhibitor specifically binds to the mutant KRAS protein, e.g., MRTX1133, sotorasib, adagrasib).
  • the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence of any one of SEQ ID NOs: 1-37, 241-257, 271, and 273-341.
  • the target sequence has an amino acid sequence of any one of SEQ ID NOs: 1-37, 241-257, and 271.
  • the target sequence has an amino acid sequence of 3, 19, or 34.
  • the guide RNA is 100% complementary to the target sequence, and/or has the same length as the target sequence.
  • the individual is resistant, refractory or recurrent to the KRAS inhibitor (e.g., MRTX1133, RMC-9805, sotorasib, adagrasib, or ganetespib).
  • the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • the individual comprises a secondary mutation in KRAS, optionally wherein the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation.
  • the cancer comprises a copy number variation in KRAS.
  • the cancer comprises an upregulated KRAS mRNA level and/or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • the cellpenetrating peptide comprises a linker between the targeting moiety and the amino acid sequence of any one of SEQ ID Nos: 137, 138, 89 and 355.
  • the linker comprises a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3, Aun, Ava, and Ahx.
  • the CPP comprises an amino acid sequence of any one of SEQ ID Nos: 96, 261, 272, 162, 353, and 354.
  • the complex comprises a second CPP.
  • the second CPP is selected from the group consisting of SEQ ID Nos: 137, 138, 89 and 355.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 96, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence of SEQ ID NO: 34, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of sotorasib and adagrasib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 162, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 34, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of sotorasib and adagrasib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 353, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 34, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of sotorasib and adagrasib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 354, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 34, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of sotorasib and adagrasib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 96, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 34, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of sotorasib and adagrasib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 162, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 34, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of sotorasib and adagrasib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 353, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 34, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of sotorasib and adagrasib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 354, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 34, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of sotorasib and adagrasib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 96, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 19, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of MRTX1133 and RMC-9805.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 162, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 19, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of MRTX1133 and RMC-9805.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 353, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 19, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of MRTX1133 and RMC-9805.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 354, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 19, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of MRTX1133 and RMC-9805.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 96, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 19, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of MRTX1133 and RMC-9805.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 162, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 19, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of MRTX1133 and RMC-9805.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 353, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 19, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of MRTX1133 and RMC-9805.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 354, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 19, wherein the individual has been subjected to a KRAS inhibitor selected from the group consisting of MRTX1133 and RMC-9805.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a noncancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 96, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 3, wherein the individual has been subjected to a KRAS inhibitor consisting of ganetespib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 162, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 3, wherein the individual has been subjected to a KRAS inhibitor consisting of ganetespib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 353, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 3, wherein the individual has been subjected to a KRAS inhibitor consisting of ganetespib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 154, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 354, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 3, wherein the individual has been subjected to a KRAS inhibitor consisting of ganetespib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 96, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 3, wherein the individual has been subjected to a KRAS inhibitor consisting of ganetespib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 162, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 3, wherein the individual has been subjected to a KRAS inhibitor consisting of ganetespib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 353, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 3, wherein the individual has been subjected to a KRAS inhibitor consisting of ganetespib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising 1) a first cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 90, 2) a second cell-penetrating peptide comprising an amino acid sequence of SEQ ID NO: 354, 3) an mRNA encoding Cas9, and 4) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence consisting of SEQ ID NO: 3, wherein the individual has been subjected to a KRAS inhibitor consisting of ganetespib.
  • crRNA specificity-determining CRISPR RNA
  • the individual does not develop a secondary KRAS mutation in any of the exons.
  • the cancer comprises an upregulated KRAS transcript level.
  • the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter.
  • the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the increased wildtype RAS signaling comprises an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and/or N-RAS.
  • a molar ratio of the cell-penetrating peptide to the mRNA to the guide RNA is between about 10:1:1 and about 25: 1 : 1.
  • Kirsten rat sarcoma (KRAS) gene belongs to a member of the RAS family and its mutations are genetic drivers of multiple cancer types, especially colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC).
  • KRAS- 012 mutations (89%) predominate in human cancers, followed by G13 (9%) and Q61 (1%) mutations.
  • the G12D mutation is the most common mutation among three common G12C (14%), G12D (36%), and G12V (23%) mutations.
  • G12C is the most common mutation subtype in NSCLC (13%). Identifying specific types of KRAS mutations in combination with other gene mutations may provide information about disease aggressiveness or drug sensitivity, which is the basis of precision medicine or personalized care.
  • KRAS protein is a signaling GTPase that switches between the active GTP-bound and inactive GDP-bound conformations.
  • Guanine nucleotide exchange factors GEF
  • GAP GTPase-activating proteins
  • RTKs receptor tyrosine kinases
  • SOS Ras/Rac guanine nucleotide exchange factor 1 S0S1
  • PTPN11 protein tyrosine phosphatase non-receptor type 11
  • SHP2 protein tyrosine phosphatase non-receptor type 11
  • switch-I and switch-II Comparing the GTP- and GDP-bound structures of KRAS identified two regions, called switch-I and switch-II.
  • the mutant cysteine 12 is located next to the pocket (P2) in the switch-II region.
  • the KRAS mutation disrupts the guanine exchange cycle, thereby locking it in an inactive GDP- bound form that drives pro-tumorigenic signals.
  • the oncogenic KRAS signal establishes the main signal axis of tumor cell proliferation and survival, providing a key target for cancer treatment.
  • KRAS-G12C inhibitors namely sotorasib (also known as AMG 510 or Lumakras) and adagrasib (also known as MRTX849), which act by selectively forming a covalent bond with cysteine 12 within the switch-II pocket of KRAS-G12C protein, thereby locking KRAS in the inactive state to arrest cell proliferation.
  • sotorasib also known as AMG 510 or Lumakras
  • adagrasib also known as MRTX849
  • sotorasib and adagrasib selectively impair the viability of KRAS-G12C mutant cell lines, but do not affect cell lines with other KRAS mutations in vitro and in vivo. Both sotorasib and adagrasib have long half-life (5.5-24 h) and extensive tissue distribution in human. Unexpectedly, neither sotorasib nor adagrasib affects PI3K signaling, indicating that the upstream pathway independent of KRAS-G12C facilitates the activation of PI3K, which provides an explanation for the formation of KRAS-G12C inhibitor resistance.
  • NCT04330664 Clinical trials (NCT04330664) are ongoing to test the combination with TNO155 (SHP2 inhibitor) and adagrasib in patients with advanced solid tumors carrying KRAS-G12C mutation.
  • TNO155 SHP2 inhibitor
  • adagrasib activation of the PI3K-AKT-mT0R pathway contributes to the development of sotorasib resistance in human PDAC cell line in vitro and in xenograft mouse models.
  • Gene set enrichment analysis and mass spectrometry-based phosphoproteomics analysis found that induction of epithelial-to- mesenchymal transition (EMT) promotes resistance to sotorasib or ARS-1620 through activation of PI3K or ERK pathway in NSCLC cells in a cell type-dependent manner.
  • EMT epithelial-to- mesenchymal transition
  • Nuclear factor, erythroid 2 like 2 (NFE2L2, best known as NRF2) regulated by kelch like ECH associated protein 1 (KEAP1) is a key transcription factor in cellular antioxidant response.
  • KEAP1 or NFE2L2 mutations that predict poor response to checkpoint inhibitor immunotherapy may also be related to resistance to adagrasib.
  • intrinsic or adaptive resistance may be caused by concurrent genetic changes, such as secondary KRAS mutations and other genetic mutations, which are not targeted by KRAS-G12C inhibitors.
  • KRAS-G12C inhibitors include glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, glutamate, Y, Y-G12C inhibitors.
  • next-generation sequencing and deep mutation scanning to characterize genetic variations in tissue samples or circulating tumor DNA from patients resistant to adagrasib.
  • 45% patients are found a putative resistance mechanism to adagrasib.
  • KRAS- Y96D mutation directly affects the binding of adagrasib to the P2 pocket, thereby conferring resistance to sotorasib, adagrasib, or ARS-1620 in multiple cancer cell lines (H358, MIAPaCa2, and BaF3).
  • RM-018 a representative KRAS-G12C-selective inhibitor from Revolution Medicines’ new class of RAS(ON) inhibitors, retains potent inhibitory activity against tumor cells harboring dual KRAS-G12C/Y92D mutations.
  • KRAS-Y96D KRAS-Y96S and KRAS-Y96C also contribute to the resistance to sotorasib or adagrasib in BaF3 cells, and this process can be reversed by the combined use of SOS1 inhibitors (B 1-3406).
  • SOS1 inhibitors B 1-3406
  • the prior KRAS inhibitor is a small molecular inhibitor. In some embodiments, the prior KRAS inhibitor is an inhibitor specifically targeting a mutation on codon 12. In some embodiments, the KRAS inhibitor inhibits specific KRAS mutation selected from the group consisting of G12C, G12D, and G12V. In some embodiments, the KRAS inhibitor covalently binds to the mutant cysteine and traps the mutant KRAS in an inactive form. In some embodiments, the KRAS inhibitor utilizes cyclophilin A to block GTP-bound KRASG12C from interacting with downstream molecules in the signaling pathway.
  • the KRAS inhibitor binds to a pocket of the switch II region of inactive GDP-form of KRASG12C mutant protein. In some embodiments, the KRAS inhibitor locks the KRASG12C mutant protein in an inactive GDP-bound conformation. In some embodiments, the KRAS inhibitor inhibits the activity of heat-shock protein 90.
  • the KRAS inhibitor is a G12C inhibitor.
  • G12C inhibitors can be found in e.g., J Exp Clin Cancer Res 41, 27 (2022), which in incorporated herein in its entirety.
  • the KRAS inhibitor is ARS-1620, RM-018, sotorasib or adagrasib.
  • the KRAS inhibitor is a G12D inhibitor.
  • G12D inhibitors can be found in e.g., Cell Discov 8, 5 (2022), which in incorporated herein in its entirety.
  • the G12D inhibitor is MRTX1133 or RMC-9805.
  • the KRAS inhibitor is a G12V inhibitor.
  • Exemplary G12V inhibitors can be found in e.g., Cancer Res (2021) 81 (13_Supplement): 1260, which in incorporated herein in its entirety.
  • the G12V inhibitor is ganetespib.
  • the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • the complex is administered no more than about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • Mutant KRAS e.g., mutation on codon 12, e.g., secondary mutation post prior KRAS inhibitor treatment
  • the cancer tissue has a KRAS aberration.
  • the aberration of KRAS comprises a mutation on codon 12.
  • the mutation on codon 12 is a somatic mutation.
  • the mutation on codon 12 is a germline mutation.
  • the aberration of KRAS is selected from the group consisting of G12C, G12D, G12V, G12R, G12A, and G12S.
  • the aberration of KRAS is G12C, G12D and/or G12V.
  • the cancer tissue has a KRAS mutation on codon 13, 18, 59, 61, 68, 95, 96, 117, or 146.
  • the cancer tissue has a G13C/D, A18D, Q61H/L, A59T, R68S/M, H95D/Q/R, Y96C/D, K117N, and/or A146T mutation.
  • any one of the mutations on codon 13, 18, 59, 61, 68, 95, 96, 117, and/or 146 are germline mutation.
  • any one of the mutations on codon 13, 18, 59, 61, 68, 95, 96, 117, and/or 146 are somatic mutation.
  • the cancer tissues has a secondary mutation in KRAS after the prior KRAS inhibitor treatment.
  • the cancer tissue developed a KRAS mutation on codon 13, 18, 59, 61, 68, 95, 96, 117, or 146 after the prior KRAS inhibitor treatment.
  • the cancer tissue developed a G13C/D, A18D, Q61H/L, A59T, R68S/M, H95D/Q/R, Y96C/D, KI 17N, and/or A146T mutation after the prior KRAS inhibitor treatment.
  • the cancer tissue developed a R68M, Y96D, or A59T mutation after the prior KRAS inhibitor treatment.
  • KRAS genetic aberrations of KRAS refers to a genetic aberration, an aberrant expression level and/or an aberrant activity level of one or more KRAS-associated gene that may lead to hyperactivation of the KRAS signaling pathway.
  • “Hyperactivate” refers to increase of an activity level of a molecule (such as a protein or protein complex) or a signaling pathway (such as the RAS/MAPK pathway) to a level that is above a reference activity level or range, such as at least about any of 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 100%, 200%, 500% or more above the reference activity level or the median of the reference activity range.
  • the reference activity level is a clinically accepted normal activity level in a standardized test, or an activity level in a healthy individual (or tissue or cell isolated from the individual) free of an KRAS -activating aberration.
  • the KRAS-activating aberration contemplated herein may include one type of aberration at one KRAS-associated gene, more than one type (such as at least about any of 2, 3, 4, 5, 6, or more) of aberrations in one KRAS-associate gene, one type of aberration at more than one (such as at least about any of 2, 3, 4, 5, 6, or more) KRAS-associated genes, or more than one type (such as at least about any of 2, 3, 4, 5, 6, or more) of aberration at more than one (such as at least about any of 2, 3, 4, 5, 6, or more) KRAS-associated genes.
  • Different types of KRAS-activating aberration may include, but are not limited to, genetic aberrations, aberrant expression levels (e.g.
  • a genetic aberration comprises a change to the nucleic acid (such as DNA or RNA) or protein sequence (i.e. mutation) or an aberrant epigenetic feature associated with a KRAS-associated gene, including, but not limited to, coding, non-coding, regulatory, enhancer, silencer, promoter, intron, exon, and untranslated regions of the KRAS-associated gene.
  • the KRAS-activating aberration comprises a mutation of an KRAS-associated gene, including, but not limited to, deletion, frameshift, insertion, indel, missense mutation, nonsense mutation, point mutation, silent mutation, splice site mutation, splice variant, and translocation.
  • the genetic aberration comprises a copy number variation of an KRAS-associated gene.
  • the copy number variation of the KRAS-associated gene is caused by structural rearrangement of the genome, including deletions, duplications, inversion, and translocations.
  • the genetic aberration comprises an aberrant epigenetic feature of an KRAS-associated gene, including, but not limited to, DNA methylation, hydroxymethylation, increased or decreased histone binding, chromatin remodeling, and the like.
  • the KRAS-activating aberration is determined in comparison to a control or reference, such as a reference sequence (such as a nucleic acid sequence or a protein sequence), a control expression (such as RNA or protein expression) level, a control activity (such as activation or inhibition of downstream targets) level, or a control protein phosphorylation level.
  • a control or reference such as a reference sequence (such as a nucleic acid sequence or a protein sequence), a control expression (such as RNA or protein expression) level, a control activity (such as activation or inhibition of downstream targets) level, or a control protein phosphorylation level.
  • the aberrant expression level or the aberrant activity level in a KRAS- associated gene may be above the control level (such as about any of 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 100%, 200%, 500% or more above the control level) if the KRAS- associated gene is a positive regulator (i.e.
  • control level e.g. expression level or activity level
  • the control level is the median level (e.g. expression level or activity level) of a control population.
  • the control population is a population having the same cancer as the individual being treated.
  • the control population is a healthy population that does not have the cancer, and optionally with comparable demographic characteristics (e.g. gender, age, ethnicity, etc.) as the individual being treated.
  • control level is a level (e.g. expression level or activity level) of a healthy tissue from the same individual.
  • a genetic aberration may be determined by comparing to a reference sequence, including epigenetic patterns of the reference sequence in a control sample.
  • the reference sequence is the sequence (DNA, RNA or protein sequence) corresponding to a fully functional allele of an KRAS-associated gene, such as an allele (e.g. the prevalent allele) of the KRAS-associated gene present in a healthy population of individuals that do not have the cancer, but may optionally have similar demographic characteristics (such as gender, age, ethnicity etc.) as the individual being treated.
  • the genetic aberrations of KRAS may be assessed based on a sample, such as a sample from the individual and/or reference sample.
  • the sample is a tissue sample or nucleic acids extracted from a tissue sample.
  • the sample is a cell sample (for example a CTC sample) or nucleic acids extracted from a cell sample.
  • the sample is a tumor biopsy.
  • the sample is a tumor sample or nucleic acids extracted from a tumor sample.
  • the sample is a biopsy sample or nucleic acids extracted from the biopsy sample.
  • the sample is a Formaldehyde Fixed-Paraffin Embedded (FFPE) sample or nucleic acids extracted from the FFPE sample.
  • the sample is a blood sample.
  • cell-free DNA is isolated from the blood sample.
  • the biological sample is a plasma sample or nucleic acids extracted from the plasma sample.
  • the genetic aberrations of KRAS may be determined by any method known in the art. See, for example, Dickson et al. Int. J. Cancer, 2013, 132(7): 1711-1717; Wagle N. Cancer Discovery, 2014, 4:546-553; and Cancer Genome Atlas Research Network. Nature 2013, 499: 43-49.
  • Exemplary methods include, but are not limited to, genomic DNA sequencing, bisulfite sequencing or other DNA sequencing-based methods using Sanger sequencing or next generation sequencing platforms; polymerase chain reaction assays; in situ hybridization assays; and DNA microarrays.
  • the epigenetic features (such as DNA methylation, histone binding, or chromatin modifications) of one or more genes from a sample isolated from the individual may be compared with the epigenetic features of the one or more genes from a control sample.
  • the nucleic acid molecules extracted from the sample can be sequenced or analyzed for the presence of the genetic aberrations relative to a reference sequence, such as the wildtype sequences of KRAS.
  • the genetic aberration of KRAS is assessed using cell-free DNA sequencing methods. In some embodiments, the genetic aberration of KRAS is assessed using next-generation sequencing. In some embodiments, the genetic aberration of KRAS isolated from a blood sample is assessed using next-generation sequencing. In some embodiments, the genetic aberration of KRAS is assessed using exome sequencing. In some embodiments, the genetic aberration of KRAS is assessed using fluorescence in-situ hybridization analysis. In some embodiments, the genetic aberration of KRAS is assessed prior to initiation of the methods of treatment described herein. In some embodiments, the genetic aberration of KRAS is assessed after initiation of the methods of treatment described herein. In some embodiments, the genetic aberration of KRAS is assessed prior to and after initiation of the methods of treatment described herein. An aberrant level of KRAS may refer to an aberrant expression level or an aberrant activity level.
  • Secondary mutations refer to additional mutations that develop in response to small molecule inhibitors. Secondary mutations can be determined by the methods described herein.
  • the individual does not develop a secondary KRAS mutation in any of the exons after the KRAS inhibitor treatment. In some embodiments, the individual does not develop a secondary KRAS mutation in a full-length KRAS gene sequence and/or promoter region of KRAS.
  • a method of treating a cancer in an individual comprising administering to the individual a complex comprising: a-1) a cell-penetrating peptide, and a-2) a guide RNA targeting a mutated KRAS, comprising a specificitydetermining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1- 37, 241-257, 271, and 273-341, wherein the individual does not develop a secondary KRAS mutation in any of the exons after the KRAS treatment.
  • crRNA specificitydetermining CRISPR RNA
  • the individual is resistant to a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-9805, or ganetespib).
  • a KRAS inhibitor e.g., sotorasib, adagrasib, MRTX1133, RMC-9805, or ganetespib.
  • the individual harbors an additional KRAS aberration other than the specific mutation targeted by the specific guide RNA in the complex (e.g., G12C or G12D).
  • the individual has an abnormal RAS pathway (e.g., overexpressed H-RAS and/or N-RAS protein).
  • the disease is a cancer. In some embodiments, the diseases is myelodysplastic syndrome.
  • the cancer is a leukemia or lymphoma. In some embodiments, the cancer is a solid tumor.
  • the solid tumor includes, but is not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, Kaposi's sarcoma, soft tissue sarcoma, uterine sacronomasynovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcino
  • the disease is selected from the group consisting of myelodysplastic syndrome, lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • lung cancer e.g., NSCLC, small cell lung cancer, squamous cell lung cancer
  • the cancer is pancreatic cancer (e.g., pancreatic ductal adenocarcinoma) .
  • the cancer is colorectal cancer.
  • the cancer is lung cancer (e.g., NSCLC).
  • the cancer is a malignant and/or advanced cancer.
  • combination therapies for treating a disease comprising: a) administering into the individual a complex or nanoparticle described herein, and b) administering to the individual a second agent or therapy.
  • the second agent described herein can be any medication or therapy that is useful for treating the disease (such as a standard therapy for the disease).
  • the second agent comprises a chemotherapeutic agent.
  • the second agent comprises a taxane.
  • the second agent comprises a cytotoxic nucleoside analogue.
  • the complex or nanoparticle composition and/or the second agent/therapy are administered simultaneously. In some embodiments, the complex or nanoparticle composition and/or the second agent/therapy are administered sequentially. In some embodiments, the complex or nanoparticle composition and/or the second agent/therapy are administered concurrently.
  • the dosing frequency of the complex or nanoparticle composition and/or the second agent/therapy may be adjusted over the course of the treatment, based on the judgment of the administering physician.
  • the complex or nanoparticle composition and/or the second agent/therapy can be administered at different dosing frequency or intervals.
  • sustained continuous release formulation of the complex or nanoparticle composition and/or the second agent/therapy may be used.
  • formulations and devices for achieving sustained release are known in the art.
  • a combination of the administration configurations described herein can also be used.
  • the complex or nanoparticle composition is administered to the individual at a frequency of about twice a week to about once every two weeks (e.g., about once a week). In some embodiments, the complex or nanoparticle composition is administered to the individual at least twice.
  • the complex or nanoparticle composition and/or the second agent/therapy can be administered using the same route of administration or different routes of administration.
  • the complex or second agent/therapy described herein is administered to the individual by any of intravenous, intratumoral, intraarterial, topical, intraocular, ophthalmic, intraportal, intracranial, intracerebral, intracerebroventricular, intrathecal, intravesicular, intradermal, subcutaneous, intramuscular, intranasal, intratracheal, pulmonary, intracavity, or oral administration, or nebulization (NB) or intratracheal instillation.
  • NB nebulization
  • the complex or nanoparticle composition and/or the second agent/therapy as described herein is formulated for systemic or tropical administration.
  • the complex or nanoparticle composition and/or the second agent/therapy as described herein is formulated for intravenous, intratumoral, intraarterial, topical, intraocular, ophthalmic, intraportal, intracranial, intracerebral, intracerebroventricular, intrathecal, intravesicular, intradermal, subcutaneous, intramuscular, intranasal, intratracheal, pulmonary, intracavity, or oral administration, or nebulization (NB) or intratracheal instillation.
  • NB nebulization
  • dosages of the guide RNA or the total nucleic acid in the cargo are in the range of about 0.001 mg/kg to about 100 mg/kg for each administration.
  • the exemplary dosage the guide RNA or the total nucleic acid in the cargo is about 0.005mg/kg to about 0.5 mg/kg (e.g., about O.Olmg/kg to about 0.05mg/kg, about 0.02mg/kg to about 0.04mg/kg) for each administration in the individual.
  • the individual is a human being.
  • dosages of the guide RNA or the total nucleic acid in the cargo are in the range of about 0.01 mg/m2 to about 1000 mg/m2 for each administration.
  • the exemplary dosage of the guide RNA or the total nucleic acid in the cargo is about 0.01 mg/m2 to about 50 mg/m2 (e.g., about 0.1 mg/m2 to about 5 mg/m2, about 0.5 mg/m2 to about 3 mg/m2) for each administration in the individual.
  • the individual is a human being.
  • the doses required for the guide RNA or the second agent/therapy may (but not necessarily) be lower than what is normally required when each agent is administered alone.
  • a subtherapeutic amount of the guide RNA or the second agent/therapy is administered, “subtherapeutic amount” or “subtherapeutic level” refer to an amount that is less than the therapeutic amount, that is, less than the amount normally used when the drug in the nanoparticle composition and/or the other agent are administered alone.
  • the reduction may be reflected in terms of the amount administered at a given administration and/or the amount administered over a given period of time (reduced frequency).
  • the dose of both the guide RNA or the second agent/therapy are reduced as compared to the corresponding normal dose of each when administered alone.
  • the guide RNA or the second agent/therapy are administered at a subtherapeutic, i.e., reduced, level.
  • the dose of guide RNA or the second agent/therapy is substantially less than the established maximum toxic dose (MTD).
  • the dose of the guide RNA or the second agent/therapy is less than about 50%, 40%, 30%, 20%, or 10% of the MTD.
  • a combination of the administration configurations described herein can be used.
  • the methods described herein may be performed alone or in conjunction with another therapy, such as chemotherapy, radiation therapy, surgery, hormone therapy, gene therapy, immunotherapy, chemoimmunotherapy, hepatic artery-based therapy, cryotherapy, ultrasound therapy, liver transplantation, local ablative therapy, radiofrequency ablation therapy, photodynamic therapy, and the like.
  • a person having a greater risk of developing a cancer may receive treatments to inhibit or and/or delay the development of the disease.
  • compositions described herein allow infusion of the composition to an individual over an infusion time that is shorter than about 24 hours.
  • the composition is administered over an infusion period of less than about any of 24 hours, 12 hours, 8 hours, 5 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes.
  • the composition is administered over an infusion period of about 30 minutes.
  • Complexes e.g., genome-editing complexes, complexes comprising a RNAi
  • the complex described herein comprises a genome-editing complex.
  • the genome-editing complex comprising a cell-penetrating peptide (such as any of the CPPs described herein), a DNA nuclease, and a polynucleotide comprising any of the guide RNA described herein.
  • the genome-editing complex comprises a) a first cellpenetrating peptide, and b) a polynucleotide comprising a guide RNA targeting mutated KRAS comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, and 273-341.
  • the complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding the DNA nuclease.
  • the DNA nuclease is selected from the group consisting of a CRISPR-associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof.
  • the guide RNA comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34.
  • the first CPP comprises from N-terminus to C- terminus a) a targeting moiety comprising an amino acid sequence of any one of 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426 (e.g., SEQ ID NO: 201, 351, or 352), and b) an amino acid sequence of any one of SEQ ID Nos: 137, 138, 89 and 355.
  • the target sequence has an amino acid sequence of 3, 19, or 34.
  • the guide RNA is 100% complementary to the target sequence, and/or has the same length as the target sequence.
  • the cell-penetrating peptide comprises a linker between the targeting moiety and the amino acid sequence of any one of SEQ ID Nos: 137, 138, 89 and 355.
  • the linker comprises a polyglycine linker moiety, a PEG moiety (e.g., (PEG)3, Aun, Ava, and Ahx.
  • the CPP comprises an amino acid sequence of any one of SEQ ID Nos: 96, 261, 272, and 162.
  • the complex comprises a second CPP.
  • the second CPP is selected from the group consisting of SEQ ID Nos: 137, 138, 89 and 355.
  • the complex comprises a) a first cell-penetrating peptide, and b) a guide RNA targeting KRAS G12V comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8.
  • the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, and 8.
  • the target sequence is set forth in SEQ ID NO: 3.
  • the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cellpenetrating peptide.
  • the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the first cell-penetrating peptide further comprises a carbohydrate moiety (such as GalNAc). In some embodiments, the first cellpenetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide is a VEPEP-3 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-62. In some embodiments, the first cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270. In some embodiments, the first cell-penetrating peptide is a VEPEP-9 peptide. In some embodiments, the first cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134.
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2: 1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1).
  • the guide RNA is complexed with the first cell-penetrating peptide.
  • the complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding the DNA nuclease.
  • the complex comprises a) a first cell-penetrating peptide, and b) a guide RNA targeting KRAS G12D comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23.
  • the target sequence is selected from the group consisting of SEQ ID NOs: 16, 19-21, and 23.
  • the target sequence is selected from the group consisting of SEQ ID NO: 19.
  • the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide.
  • the first cellpenetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Ann, Ava, and Ahx. In some embodiments, the first cell-penetrating peptide further comprises a carbohydrate moiety (such as GalNAc). In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide is a VEPEP-3 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-62. In some embodiments, the first cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270. In some embodiments, the first cell-penetrating peptide is a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134.
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1).
  • the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding the DNA nuclease.
  • a DNA nuclease e.g., Cas9
  • the complex comprises a) a first cell-penetrating peptide, and b) a guide RNA targeting KRAS G12C comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34.
  • the target sequence is set forth in SEQ ID NO: 34.
  • the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cellpenetrating peptide.
  • the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the first cell-penetrating peptide further comprises a carbohydrate moiety (such as GalNAc). In some embodiments, the first cellpenetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide is a VEPEP-3 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-62. In some embodiments, the first cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270. In some embodiments, the first cell-penetrating peptide is a VEPEP-9 peptide. In some embodiments, the first cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134.
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2: 1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1).
  • the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding the DNA nuclease.
  • a DNA nuclease e.g., Cas9
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is an ADGN-100 peptide; and b) a guide RNA targeting a mutated KRAS comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 153-175. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • tracrRNA auxiliary trans-activating crRNA
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1).
  • the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding the DNA nuclease.
  • a DNA nuclease e.g., Cas9
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is a VEPEP-6 peptide; and b) a guide RNA targeting a mutated KRAS comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (such as between about 5: 1 and about 20:1, such as between about 2: 1 to about 50:1).
  • the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding the DNA nuclease.
  • a DNA nuclease e.g., Cas9
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is a VEPEP-9 peptide; and b) a guide RNA targeting a mutated KRAS comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134.
  • the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (such as between about 5: 1 and about 20:1, such as between about 2: 1 to about 50:1).
  • the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1).
  • the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1).
  • the guide RNA is complexed with the first cell-penetrating peptide.
  • the guide RNA is complexed with the first cell-penetrating peptide.
  • the complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding the DNA nuclease.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide; b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease) or a polynucleotide encoding the DNA nuclease.
  • a DNA nuclease e.g., a CRISPR-associated endonuclease
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide; b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease) or a polynucleotide encoding the DNA nuclease.
  • a DNA nuclease e.g., a CRISPR-associated endonuclease
  • the first cell-penetrating peptide is an ADGN-100 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (such as an amino acid sequence selected from the group consisting of 153-175, such as an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172).
  • the first cell-penetrating peptide comprises a VEPEP-6 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270.
  • the first cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cell-penetrating peptide comprises a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118- 134. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9).
  • the DNA nuclease is a fusion protein, wherein the fusion protein further comprises a second enzyme that will allow base editing or prime editing.
  • the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme.
  • the one or more guide RNA comprise at least two guide RNA that specifically target at least two different KRAS mutations selected from G12V, G12D, and G12C.
  • the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1: 1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides; and b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NO: 3.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides; b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NO: 3; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., a Cas polypeptide, e.g., Cas9 or Cas 12a) or a polynucleotide encoding the DNA nuclease.
  • a DNA nuclease e.
  • the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (such as an amino acid sequence selected from the group consisting of 153-175, such as an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172). In some embodiments, the first cell-penetrating peptide comprises a VEPEP-6 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270. In some embodiments, the first cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cell-penetrating peptide comprises a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118- 134. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • tracrRNA auxiliary trans-activating crRNA
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80: 1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides; and b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence set forth in any one of SEQ ID Nos: 273-341.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides; b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NO: 3; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., a Cas polypeptide, e.g., Cas9 or Cas 12a) or a polynucleotide encoding the DNA nuclease.
  • a DNA nuclease e.
  • the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide is an ADGN- 100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (such as an amino acid sequence selected from the group consisting of 153-175, such as an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172). In some embodiments, the first cell-penetrating peptide comprises a VEPEP-6 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cellpenetrating peptide comprises a VEPEP-9 peptide. In some embodiments, the first cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • tracrRNA auxiliary trans-activating crRNA
  • the molar ratio of the first cellpenetrating peptide to the guide RNA is between about 1:1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides; and b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NO: 19.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides; b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NO: 19; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., a Cas polypeptide, e.g., Cas9 or Cas 12a) or a polynucleotide encoding the DNA nuclease.
  • a DNA nuclease e.
  • the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (such as an amino acid sequence selected from the group consisting of 153-175, such as an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172). In some embodiments, the first cell-penetrating peptide comprises a VEPEP-6 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270. In some embodiments, the first cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cell-penetrating peptide comprises a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118- 134. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • tracrRNA auxiliary trans-activating crRNA
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80: 1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides; and b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NO: 34.
  • the complex comprises a) a first cell-penetrating peptide, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, LNCOV peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides; b) one or more guide RNA targeting a mutated KRAS comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NO: 34; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., a Cas polypeptide, e.g., Cas9 or Cas 12a) or a polynucleotide encoding the DNA nuclease.
  • a DNA nuclease e.
  • the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (such as an amino acid sequence selected from the group consisting of 153-175, such as an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172). In some embodiments, the first cell-penetrating peptide comprises a VEPEP-6 peptide.
  • the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266 and 270. In some embodiments, the first cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cell-penetrating peptide comprises a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118- 134. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • tracrRNA auxiliary trans-activating crRNA
  • the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80: 1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide.
  • the complex comprises a RNAi (e.g., a siRNA) as described herein that specifically targets a mutant KRAS.
  • a RNAi e.g., a siRNA
  • CPP Cell Penetrating Peptides
  • CPPs can be subdivided into two main classes, the first requiring chemical linkage with the cargo and the second involving the formation of stable, non-covalent complexes.
  • CPPs from both strategies have been reported to favour the delivery of a large panel of cargos (plasmid DNA, oligonucleotide, siRNA, PNA, protein, peptide, liposome, nanoparticle%) into a wide variety of cell types and in vivo models (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206; Mickan et al. (2014) Curr Pharm Biotechnol 15, 200-209; Shukla et al. (2014) Mol Pharm 11, 3395-3408).
  • CPP Cell Penetrating Peptide
  • WO2014/053879 discloses VEPEP-3 peptides
  • WO2014/053881 discloses VEPEP-4 peptides
  • WO2014/053882 discloses VEPEP-5 peptides
  • W02012/137150 discloses VEPEP- 6 peptides
  • W02014/053880 discloses VEPEP-9 peptides
  • WO 2016/102687 discloses ADGN-100 peptides
  • US2010/0099626 discloses CADY peptides
  • U.S. Pat. No. 7,514,530 discloses MPG peptides; the disclosures of which are hereby incorporated herein by reference in their entirety.
  • the cell-penetrating peptides in the genome-editing complexes or nanoparticles of the present application are capable of forming stable complexes and nanoparticles with various molecules of a genome-editing system, such as nucleases (e.g., ZFNs, TALENs, and CRISPR-associated nucleases (such as Cas9 and Cpfl)), integrases (such as bacteriophage integrases, e.g., ⁇ I>C31), and nucleic acids (e.g., guide RNAs, guide DNAs, and donor nucleic acids).
  • nucleases e.g., ZFNs, TALENs, and CRISPR-associated nucleases (such as Cas9 and Cpfl)
  • integrases such as bacteriophage integrases, e.g., ⁇ I>C31
  • nucleic acids e.g., guide RNAs, guide DNAs, and donor nucleic acids.
  • a complex or nanoparticle described herein comprises a cellpenetrating peptide selected from the group consisting of CADY, PEP-1, MPG, LNCOV peptides, VEPEP-3 peptides, VEPEP-4 peptides, VEPEP-5 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the cell-penetrating peptide is present in a genome-editing complex.
  • the cell-penetrating peptide is present in a complex present in the core of a nanoparticle.
  • the cell-penetrating peptide is present in the core of a nanoparticle. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRIS PR-associated endonuclease, such as Cas9). In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and is associated with the guide RNA.
  • a DNA nuclease such as a CRIS PR-associated endonuclease, such as Cas9
  • the cell-penetrating peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and is associated with the guide RNA
  • the cellpenetrating peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the cell-penetrating peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the cell-penetrating peptide is present in the surface layer of a nanoparticle. In some embodiments, the cell-penetrating peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • W02022/020782 discloses LNCOV peptides; WO2014/053879 discloses VEPEP-3 peptides; WO2014/053881 discloses VEPEP-4 peptides; WO2014/053882 discloses VEPEP-5 peptides; W02012/137150 discloses VEPEP-6 peptides; W02014/053880 discloses VEPEP-9 peptides; WO 2016/102687 discloses ADGN-100 peptides; US2010/0099626 discloses CADY peptides; and.
  • U.S. Pat. No. 7,514,530 discloses MPG peptides; the disclosures of which are hereby incorporated herein by reference in their entirety.
  • a complex or nanoparticle described herein comprises a VEPEP-3 cell-penetrating peptide comprising the amino acid sequence X1X2X3X4X5X2X3X4X6X7X3X8X9X10X1 1X12X13 (SEQ ID NO: 44), wherein XI is beta-A (“beta-alanine) or S, X2 is K, R or L (independently from each other), X3 is F or W (independently from each other), X4 is F, W or Y (independently from each other), X5 is E, R or S, X6 is R, T or S, X7 is E, R, or S, X8 is none, F or W, X9 is P or R, X10 is R or L, XI 1 is K, W or R, X12 is R or F, and X13 is R or K.
  • XI beta-A (“beta-alanine) or S
  • the VEPEP-3 peptide comprises the amino acid sequence X1X2WX4EX2WX4X6X7X3PRX11RX13 (SEQ ID NO: 45), wherein XI is beta-A or S, X2 is K, R or L, X3 is F or W, X4 is F, W or Y, X5 is E, R or S, X6 is R, T or S, X7 is E, R, or S, X8 is none, F or W, X9 is P or R, X10 is R or L, XI 1 is K, W or R, X12 is R or F, and X13 is R or K.
  • the VEPEP-3 peptide comprises the amino acid sequence X1KWFERWFREWPRKRR (SEQ ID NO: 46), X1KWWERWWREWPRKRR (SEQ ID NO: 47), X1KWWERWWREWPRKRK (SEQ ID NO: 48), X1RWWEKWWTRWPRKRK (SEQ ID NO: 49), or X1RWYEKWYTEFPRRRRRR (SEQ ID NO: 50), wherein XI is beta-A or S.
  • the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, wherein the cell-penetrating peptide is modified by replacement of the amino acid in position 10 by a non-natural amino acid, addition of a non-natural amino acid between the amino acids in positions 2 and 3, and addition of a hydrocarbon linkage between the two non-natural amino acids.
  • the VEPEP-3 peptide comprises the amino acid sequence X1KX14WWERWWRX14WPRKRK (SEQ ID NO: 51), wherein XI is beta- A or S and X14 is a non-natural amino acid, and wherein there is a hydrocarbon linkage between the two non-natural amino acids.
  • the VEPEP-3 peptide comprises the amino acid sequence X1X2X3WX5X10X3WX6X7WX8X9X10WX12R (SEQ ID NO: 52), wherein XI is beta-A or S, X2 is K, R or L, X3 is F or W, X5 is R or S, X6 is R or S, X7 is R or S, X8 is F or W, X9 is R or P, X10 is L or R, and X12 is R or F.
  • the VEPEP-3 peptide comprises the amino acid sequence X1RWWRLWWRSWFRLWRR (SEQ ID NO: 53), X1LWWRRWWSRWWPRWRR (SEQ ID NO: 54), X1LWWSRWWRSWFRLWFR (SEQ ID NO: 55), or X1KFWSRFWRSWFRLWRR (SEQ ID NO: 56), wherein XI is beta-A or S.
  • the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 44 and 52-56, wherein the cell-penetrating peptide is modified by replacement of the amino acids in position 5 and 12 by non-natural amino acids, and addition of a hydrocarbon linkage between the two non-natural amino acids.
  • the VEPEP-3 peptide comprises the amino acid sequence X1RWWX14LWWRSWX14RLWRR (SEQ ID NO: 57), wherein XI is a beta-alanine or a serine and X14 is a non-natural amino acid, and wherein there is a hydrocarbon linkage between the two non-natural amino acids.
  • the VEPEP-3 peptide comprises the amino acid sequence beta- AKWFERWFREWPRKRR (SEQ ID NO: 58). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence beta-AKWWERWWREWPRKRR (SEQ ID NO: 59). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence ASSLNIA- Ava-KWWERWWREWPRKRR (SEQ ID NO: 60). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence LSSRLDA-Ava-KWWERWWREWPRKRR (SEQ ID NO: 61).
  • the VEPEP-3 peptide comprises the amino acid sequence Ac-SYTSSTM-ava-KWWERWWREWPRKRR (SEQ ID NO: 62). In some embodiments, the VEPEP-3 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-3 peptide is present in a complex in the core of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and is associated with a guide RNA.
  • the VEPEP-3 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-3 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-3 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • a complex or nanoparticle described herein comprises a VEPEP-6 cell-penetrating peptide.
  • the VEPEP-6 peptide comprises an amino acid sequence of SEQ ID NO: 355.
  • the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LX2RALWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 63), X1LX2LARWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 64) and X1LX2ARLWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 65), wherein XI is beta-A or S, X2 is F or W, X3 is L, W, C or I, X4 is S, A, N or T, X5 is L or W
  • the VEPEP-6 peptide comprises the amino acid sequence X1LX2RALWRLX3RX4LWRLX5X6X7X8 (SEQ ID NO: 66), wherein XI is beta-A or S, X2 is F or W, X3 is L, W, C or I, X4 is S, A, N or T, X5 is L or W, X6is W or R, X7 is K or R, and X8 is A or none.
  • the VEPEP-6 peptide comprises the amino acid sequence X1LX2RALWRLX3RX4LWRLX5X6KX7 (SEQ ID NO: 67), wherein XI is beta-A or S, X2 is F or W, X3 is L or W, X4 is S, A or N, X5 is L or W, X6 is W or R, X7 is A or none.
  • the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LFRALWRLLRX2LWRLLWX3 (SEQ ID NO: 68), X1LWRALWRLWRX2LWRLLWX3A (SEQ ID NO: 69), X1LWRALWRLX4RX2LWRLWRX3A (SEQ ID NO: 70), X1LWRALWRLWRX2LWRLWRX3A (SEQ ID NO: 71), X1LWRALWRLX5RALWRLLWX3A (SEQ ID NO: 72), and X1LWRALWRLX4RNLWRLLWX3A (SEQ ID NO: 73), wherein XI is beta-A or S, X2 is S or T, X3 is K or R, X4 is L, C or I and X5 is L or I.
  • the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of Ac-XILFRALWRLLRSLWRLLWK-cysteamide (SEQ ID NO: 74), Ac- XILWRALWRLWRSLWRLLWKA-cysteamide (SEQ ID NO: 75), Ac- XlLWRALWRLLRSLWRLWRKA-cysteamide (SEQ ID NO: 76), Ac- XlLWRALWRLWRSLWRLWRKA-cysteamide (SEQ ID NO: 77), Ac- XlLWRALWRLLRALWRLLWKA-cysteamide (SEQ ID NO: 78), and Ac- XlLWRALWRLLRNLWRLLWKA-cysteamide (SEQ ID NO: 79), wherein XI is beta-A or S.
  • the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 63-79, further comprising a hydrocarbon linkage between two residues at positions 8 and 12.
  • the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of Ac-XILFRALWRSLLRSSLWRLLWK- cysteamide (SEQ ID NO: 80), Ac-XILFLARWRSLLRSSLWRLLWK-cysteamide (SEQ ID NO: 81), Ac-XILFRALWSSLLRSSLWRLLWK-cysteamide (SEQ ID NO: 82), Ac- XlLFLARWSSLLRSSLWRLLWK-cysteamide (SEQ ID NO: 83), Ac- XlLFRALWRLLRSSLWSSLLWK-cysteamide (SEQ ID NO: 84), Ac- XlLFLARWRLLRSSLWSSLLWK-cysteamide (SEQ ID NO: 85), Ac- Xl
  • the VEPEP-6 peptide comprises an amino acid sequence beta-ALWRALWRLWRSLWRLLWKA (SEQ ID NO: 89). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs 90-117. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence beta- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 90). In some embodiments, the VEPEP-6 peptide comprises a retro-inverso amino acid sequence
  • the VEPEP-6 peptide comprises an amino acid sequence Ac-(PEG)7-PALWRALWRLWRSLWRLLWKA- NH2 (SEQ ID NO: 92) or Ac-(PEG)2-pALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 93). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 94-103. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence beta-A- Ac-YIGSR-Ava- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 96).
  • the VEPEP-6 peptide comprises an amino acid sequence beta-A-Ac-YIGSR-Aun- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 98). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-YIGSR-Ahx- ALWRALWRLWRSLWRLLWK-NH2 (SEQ ID NO: 100) or Ac-YIGSR-Ahx- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 101).
  • the VEPEP-6 peptide comprises an amino acid sequence beta- Ac-GYVS-Ahx- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 102) or Ac-YIGSR- PALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 103). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Stearyl-PA- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 104). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 105-107.
  • the VEPEP-6 peptide comprises an amino acid sequence ALWRA(GalNac)LWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 111). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-SYTSSTM-ava- PALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 112). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac- THRPPNWSPVWPRALWRLWRSLWRLRWKA-NH2 (SEQ ID NO: 113).
  • the VEPEP-6 peptide comprises an amino acid sequence Ac- CKTRRVPWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 114). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-CKTRRVP-ava- WRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 115). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-CARPAR-ava- WRALWRLWRSLWRLLWK-NH2 (SEQ ID NO: 116).
  • the VEPEP- 6 peptide comprises an amino acid sequence Ac-THRPPNWSPV- ava- WRALWRLWRSLWRLRWK-NH2 (SEQ ID NO: 117). In some embodiments, the VEPEP- 6 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-6 peptide is present in a complex in the core of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle.
  • the VEPEP- 6 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRIS PR-associated endonuclease, such as Cas9). In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-6 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-6 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • a complex or nanoparticle described herein comprises a VEPEP-9 cell-penetrating peptide comprising the amino acid sequence X1X2X3WWX4X5WAX6X3X7X8X9X10X11X12WX13R (SEQ ID NO: 118), wherein XI is beta- A or S, X2 is L or none, X3 is R or none, X4 is L, R or G, X5 is R, W or S, X6 is S, P or T, X7 is W or P, X8 is F, A or R, X9 is S, L, P or R, X10 is R or S, XI 1 is W or none, X12 is A, R or none and X13 is W or F, and wherein if X3 is none, then X2, XI 1 and X12 are none as well.
  • the VEPEP-9 peptide comprises the amino acid sequence X1X2RWWLRWAX6RWX8X9X10WX12WX13R (SEQ ID NO: 119), wherein XI is beta-A or S, X2 is L or none, X6 is S or P, X8 is F or A, X9 is S, L or P, X10 is R or S, X12 is A or R, and X13 is W or F.
  • the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of X1LRWWLRWASRWFSRWAWWR (SEQ ID NO: 120), X1LRWWLRWASRWASRWAWFR (SEQ ID NO: 121), X1RWWLRWASRWALSWRWWR (SEQ ID NO: 122), X1RWWLRWASRWFLSWRWWR (SEQ ID NO: 123), X1RWWLRWAPRWFPSWRWWR (SEQ ID NO: 124), and
  • the VEPEP-9 peptide comprises the amino acid sequence of X1WWX4X5WAX6X7X8RX10WWR (SEQ ID NO: 126), wherein XI is beta-A or S, X4 is R or G, X5 is W or S, X6 is S, T or P, X7 is W or P, X8 is A or R, and X10 is S or R.
  • the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of X1WWRWWASWARSWWR (SEQ ID NO: 127), X1WWGSWATPRRRWWR (SEQ ID NO: 128), and X1WWRWWAPWARSWWR (SEQ ID NO: 129), wherein XI is beta-A or S.
  • the VEPEP-9 peptide comprises the amino acid sequence beta-ALRWWLRWASRWFSRWAWWR (SEQ ID NO: 130).
  • the VEPEP-9 peptide comprises the amino acid sequence KSYDTY-ava-ALRWLRWASRWFSRWAWR (SEQ ID NO: 131).
  • the VEPEP-9 peptide comprises the amino acid sequence ac- CKRAVRWWLRWASRWFSRWAWWR (SEQ ID NO: 132). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence beta-A-RWWLRWASRWFSRWAWR (SEQ ID NO: 133). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence KSYDTYAAETRRWASRWFSRWAWWR (SEQ ID NO: 134). In some embodiments, the VEPEP-9 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-9 peptide is present in a complex in the core of a nanoparticle.
  • the VEPEP-9 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9). In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid.
  • a DNA nuclease such as a CRISPR-associated endonuclease, such as Cas9
  • the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with a gRNA.
  • the VEPEP-9 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-9 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • a complex or nanoparticle described herein comprises an ADGN-100 cell-penetrating peptide comprising the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 135), wherein XI is any amino acid or none, and X2-X8 are any amino acid.
  • the ADGN-100 peptide comprises the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 136), wherein XI is PA, S, or none, X2 is A or V, X3 is or L, X4 is W or Y, X5 is V or S, X6 is R, V, or A, X7 is S or L, and X8 is W or Y.
  • the ADGN-100 peptide comprises the amino acid sequence KWRSAGWRWRLWRVRSWSR (SEQ ID NO: 137), KWRSALYRWRLWRVRSWSR (SEQ ID NO: 138), KWRSALYRWRLWRSRSWSR (SEQ ID NO: 139), or KWRSALYRWRLWRSALYSR (SEQ ID NO: 140).
  • the ADGN-100 peptide comprises two residues separated by three or six residues that are linked by a hydrocarbon linkage.
  • the ADGN-100 peptide comprises the amino acid sequence KWRSSAGWRSWRLWRVRSWSR (SEQ ID NO: 141), KWRSSAGWRWRSLWRVRSWSR (SEQ ID NO: 142), KWRSAGWRSWRLWRVRSSWSR (SEQ ID NO: 143),
  • KWRSSALYRSWRLWRSRSWSR (SEQ ID NO: 144), KWRSSALYRWRSLWRSRSWSR (SEQ ID NO: 145), KWRSALYRSWRLWRSRSSWSR (SEQ ID NO: 146), KWRSALYRWRSLWRSSRSWSR (SEQ ID NO: 147), KWRSALYRWRLWRSSRSWSSR (SEQ ID NO: 148), KWRSSALYRWRSLWRSALYSR (SEQ ID NO: 149), KWRSSALYRSWRLWRSALYSR (SEQ ID NO: 150), KWRSALYRWRSLWRSSALYSR (SEQ ID NO: 151), or KWRSALYRWRLWRSSALYSSR (SEQ ID NO: 152), wherein the residues marked with a subscript “S” are linked by a hydrocarbon linkage.
  • the ADGN-100 peptide comprises an amino acid sequence of any one of SEQ ID NOs: 153-171. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence of beta-AKWRSAGWRWRLWRVRSWSR-NH2 (SEQ ID NO: 153). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence of beta- AKWRSAGWRWRLWRVRSWSR (SEQ ID NO: 154) or beta- AKWRSALYRWRLWRVRSWSR (SEQ ID NO: 155). In some embodiments, the ADGN- 100 peptide comprises a retro-inverso amino acid sequence of RSWSRVRWLRWRWGASRWK (SEQ ID NO: 156).
  • the ADGN- 100 peptide comprises an amino acid sequence of Ac-(PEG)7-bA- KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 157) or beta- Ac-(PEG)2-pA- KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 158).
  • the ADGN-100 peptide comprises an amino acid sequence of Stearyl-PA- KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 159).
  • the ADGN-100 peptide comprises an amino acid sequence of any one of SEQ ID NOS: 160-169.
  • the ADGN-100 peptide comprises an amino acid sequence Ac- KWRSA(GALNAC)LWRWRLWRVRSWSR-NH2 (SEQ ID NO: 172). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence Ac- CARPARWRSAGWRWRLWRVRSWSR-NH2 (SEQ ID NO: 173). In some embodiments, the ADGN-100 peptide comprises a core motif comprising an amino acid sequence of RWRLWRWSR (SEQ ID NO: 168).
  • the ADGN-100 peptide comprises an amino acid sequence TGNYKALHPDHNGWRSALRWRLWRWSR-NH2 (SEQ ID NO: 174) or Ac-TGNYKALHPDHNG-ava-WRSALRWRLWRWSR-NH2 (SEQ ID NO: 175).
  • the ADGN-100 peptide is present in a genome-editing complex.
  • the ADGN-100 peptide is present in a complex in the core of a nanoparticle.
  • the ADGN-100 peptide is present in the core of a nanoparticle.
  • the ADGN-100 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9). In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the ADGN-100 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the ADGN-100 peptide is present in the surface layer of a nanoparticle. In some embodiments, the ADGN- 100 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • a complex or nanoparticle described herein comprises a VEPEP-4 cell-penetrating peptide comprising the amino acid sequence XWXRLXXXXXX (SEQ ID NO: 176), wherein X in position 1 is beta-A or S; X in positions 3, 9 and 10 are, independently from each other, W or F; X in position 6 is R if X in position 8 is S, and X in position 6 is S if X in position 8 is R; X in position 7 is L or none; X in position 11 is R or none, and X in position 7 is L if X in position 11 is none.
  • the VEPEP- 4 peptide comprises an amino acid sequence of any one of SEQ ID NOs: 177-180. In some embodiments, the VEPEP-4 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-4 peptide is present in a complex in the core of a nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9).
  • a DNA nuclease such as a CRISPR-associated endonuclease, such as Cas9
  • the VEPEP-4 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-4 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-4 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • a complex or nanoparticle described herein comprises a VEPEP-5 cell-penetrating peptide comprising the amino acid sequence RXWXRLWXRLR (SEQ ID NO: 181), wherein X in position 2 is R or S; and X in positions 4 and 8 are, independently from each other, W or F.
  • the VEPEP-5 peptide comprises an amino acid sequence of any one of SEQ ID NOs: 182-187.
  • the VEPEP-5 peptide is present in a genome-editing complex.
  • the VEPEP-5 peptide is present in a complex in the core of a nanoparticle.
  • the VEPEP-5 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9). In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid.
  • a DNA nuclease such as a CRISPR-associated endonuclease, such as Cas9
  • the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with a gRNA.
  • the VEPEP-5 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-5 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-5 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • a complex or nanoparticle described herein comprises a LNCOV peptide (see e.g., the peptides disclosed in W02022/020782).
  • the LOCOV peptide comprises an amino acid sequence of any one of SEQ ID NOs: 356-418.
  • the LOCOV peptide is present in a genome-editing complex.
  • the LOCOV peptide is present in a complex in the core of a nanoparticle.
  • the LOCOV peptide is present in the core of a nanoparticle.
  • the LOCOV peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9). In some embodiments, the LOCOV peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the LOCOV peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the LOCOV peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the LOCOV peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the LOCOV peptide is present in the surface layer of a nanoparticle. In some embodiments, the LOCOV peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • the CPP described herein e.g., VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide
  • the CPP described herein further comprises one or more moieties linked to (e.g., covalently linked to) the N-terminus of the CPP.
  • the one or more moieties is covalently linked to the N-terminus of the CPP.
  • the one or more moieties are selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody or antibody fragment thereof, a peptide, a polysaccharide, a linker moiety, and a targeting moiety.
  • the one or more moieties comprise an acetyl group covalently linked to the N-terminus of the CPP.
  • the CPP described herein e.g., VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide
  • the CPP described herein further comprises one or more moieties linked to (e.g., covalently linked to) the C-terminus of the CPP.
  • the one or more moieties are selected from the group consisting of a cysteamide group, a cysteine, a thiol, an amide, a nitrilotriacetic acid, a carboxyl group, a linear or ramified C1-C6 alkyl group, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody or antibody fragment thereof, a peptide, a polysaccharide, a linker moiety, and a targeting moiety.
  • the one or more moieties comprises a cysteamide group.
  • the CPP described herein (e.g., PEP-1, PEP-2, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) is stapled.
  • “Stapled” as used herein refers to a chemical linkage between two residues in a peptide.
  • the CPP is stapled, comprising a chemical linkage between two amino acids of the peptide.
  • the two amino acids linked by the chemical linkage are separated by 3 or 6 amino acids.
  • two amino acids linked by the chemical linkage are separated by 3 amino acids.
  • the two amino acids linked by the chemical linkage are separated by 6 amino acids.
  • each of the two amino acids linked by the chemical linkage is R or S.
  • each of the two amino acids linked by the chemical linkage is R.
  • each of the two amino acids linked by the chemical linkage is S.
  • one of the two amino acids linked by the chemical linkage is R and the other is S.
  • the chemical linkage is a hydrocarbon linkage.
  • the CPP is an L-peptide comprising L- amino acids.
  • the CPP is a retro-inverso peptide (e.g., a peptide made up of D-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds).
  • the retro- inverso peptide comprises a sequence of SEQ ID NO: 91 or 156.
  • the CPP comprises, from N-terminus, an acetyl group, a targeting moiety and a linker moiety covalently linked to the N-terminus of the cellpenetrating peptide.
  • the one or more moieties comprise a targeting moiety.
  • the targeting moiety is conjugated to the N-terminus the CPP.
  • the targeting moiety is conjugated to the C-terminus the CPP.
  • a first targeting moiety is conjugated to the N-terminus of the CPP and a second targeting moiety is conjugated to the C-terminus of the CPP.
  • the targeting moiety comprises a targeting peptide that targets one or more organs.
  • the one or more organs are selected from the group consisting of muscle, heart, brain, spleen, lymph node, liver, lung, and kidney.
  • the targeting peptide targets brain.
  • the targeting peptide targets muscle.
  • the targeting peptide targets heart.
  • the targeting moiety comprises at least about 3, 4, or 5 amino acids. In some embodiments, the targeting moiety comprises no more than about 8, 7, 6, 5, or 4 amino acids. In some embodiments, the targeting moiety comprises about 3, 4, or 5 amino acids. In some embodiments, the targeting moiety comprises a sequence selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, IGSR. In some embodiments, the sequence (e.g., a targeting sequence) is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.
  • the targeting moiety comprises a targeting sequence selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351-352, 356-366, 378-386, 397-417, and 423-426.
  • the targeting moiety comprises a targeting sequence SYTSSTM (SEQ ID NO: 196).
  • the targeting moiety comprises a targeting sequence CKTRRVP (SEQ ID NO: 197).
  • the targeting moiety comprises a targeting sequence THRPPNWSPV (SEQ ID NO: 198).
  • the targeting moiety comprises a targeting sequence TGNYKALHPDHNG (SEQ ID NO: 199).
  • the targeting moiety comprises a targeting sequence CARPAR (SEQ ID NO: 200). In some embodiments, the targeting moiety comprises a targeting sequence ASSLNIA (SEQ ID NO: 203). In some embodiments, the targeting moiety comprises a targeting sequence LSSRLDA (SEQ ID NO: 204). In some embodiments, the targeting moiety comprises a targeting sequence KSYDTY (SEQ ID NO: 205). In some embodiments, the targeting sequence comprises the amino acid sequence of any one of KTFLDKFNHEVEDL (SEQ ID NO: 351) or YHWYGYTPQNVI (SEQ ID NO: 352). In some embodiments, the targeting sequence comprises the amino acid sequence of any one of SEQ ID Nos: 423-426. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any one of SEQ ID NOs: 353-354, and 419- 422.
  • a cell-penetrating peptide comprises from N-terminus to C- terminus: a targeting peptide, a linker moiety, and a cell-penetrating peptide.
  • the targeting peptide is covalently linked to the N-terminus of the cellpenetrating peptide.
  • the targeting sequence comprises the amino acid sequence of any one of KTFLDKFNHEVEDL (SEQ ID NO: 351) or YHWYGYTPQNVI (SEQ ID NO: 352).
  • the targeting sequence comprises the amino acid sequence of any one of SEQ ID Nos: 423-426.
  • the linker moeity is selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.
  • the cell-penetrating peptide comprises the amino acid sequence of any one of SEQ ID NOs: 353-354, and 419-422.
  • the targeting moiety is conjugated to the CPP via a linker moiety such as any one of the linker moieties described herein.
  • Linker moiety such as any one of the linker moieties described herein.
  • the one or more moieties comprise a linker moiety.
  • the linker moiety comprises a polyglycine linker.
  • the linker comprises a P-Alanine.
  • the linker comprises at least about two, three, or four glycines, optionally continuous glycines.
  • the linker further comprises a serine.
  • the linker comprises a GGGGS or SGGGG sequence.
  • the linker comprises a Glycine-P-Alanine motif.
  • the one or more moieties comprise a polymer (e.g., PEG, poly lysine, PET).
  • the polymer is conjugated to the N-terminus of the CPP.
  • the polymer is conjugated to the C-terminus of the CPP.
  • a first polymer is conjugated to the N-terminus of the CPP and a second polymer is conjugated to the C-terminus of the CPP.
  • the polymer is a PEG.
  • the PEG is a linear PEG.
  • the PEG is a branched PEG.
  • the molecular weight of the PEG is no more than about 5 kDa, 10 kDa, 15kDa, 20 kDa, 30 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is at least about 5 kDa, 10 kDa, 15kDa, 20 kDa, 30 kDa, or 40 kDa.
  • the molecular weight of the PEG is about 5 kDa to about 10 kDa, about 10 kDa to about 15kDa, about 15 kDa to about 20 kDa, about 20kDa to about 30 kDa, or about 30 kDa to about 40 kDa. In some embodiments, the molecular weight of the PEG is about 5 kDa, 10 kDa, 20 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is selected from the group consisting of 5 kDa, 10 kDa, 20 kDa or 40 kDa.
  • the molecular weight of the PEG is about 5 kDa. In some embodiments, the molecular weight of the PEG is about 10 kDa. In some embodiments, the PEG comprises at least about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG consists of no more than about 10, 9, 8 or 7 ethylene glycol units. In some embodiments, the PEG consists of about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG moiety consists of about one to eight, or about two to seven ethylene glycol units.
  • the linker moiety is selected from the group consisting of beta alanine, cysteine, cysteamide bridge, poly glycine (such as G2 or G4), Aun (11-amino- undecanoic acid), Ava (5-amino pentanoic acid), and Ahx (aminocaproic acid).
  • the linker moiety comprises Aun (11-amino-undecanoic acid).
  • the linker moiety comprises Ava (5-amino pentanoic acid).
  • the linker moiety comprises Ahx (aminocaproic acid).
  • the cell-penetrating peptide further comprises a carbohydrate moiety.
  • the carbohydrate moiety is GalNAc.
  • the cell-penetrating peptide is an ADGN-106 peptide.
  • the cellpenetrating peptide is an ADGN-100 peptide.
  • the carbohydrate moiety modifies an alanine within the cell-penetrating peptide.
  • the cellpenetrating peptide is set forth in SEQ ID NO: 111 or 172.
  • the cell-penetrating peptide in the genome-editing complexes is a mixture of a) a first peptide comprising a first cell-penetrating peptide (such as any of the cell-penetrating peptide described herein); b) a second peptide comprising a second cellpenetrating peptide (such as any of the cell-penetrating peptide described herein), wherein the second peptide comprises a polyethylene glycol (PEG) moiety that is covalently linked to the second cell-penetrating peptide, and wherein the first peptide does not have a PEG moiety.
  • PEG polyethylene glycol
  • the first and/or the second cell-penetrating peptide is a PTD-based peptide, an amphipathic peptide, a poly-arginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide.
  • the first and the second cell-penetrating peptides are selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-4 peptides, VEPEP-5 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • the molar ratio of the cell-penetrating peptide to the cargo is between about 1:1 and about 100:1 (such as about between about 1:1 and about 50:1, or about 2:1 to about 50:1).
  • the average diameter of the complex is between about 20 nm and about 1000 nm (such as about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm).
  • the PEG moiety consists of about one to ten (such as about 1-8, 2-7, 1-5, or 6-10) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa to about 0.5 kDa (such as about 0.05-0.1, 0.05-
  • the PEG moiety is conjugated to the N- or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.
  • the ratio of the first cell-penetrating peptide to the second cellpenetrating peptide is about 20:1 to about 1:1 (such as about 15:1 to about 2:1, about 10:1 to about 4:1).
  • the first and/or the second cell-penetrating peptides are selected from VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first and/or the second cell-penetrating peptide are selected from VEPEP-6 peptides, and ADGN-100 peptides.
  • the PEG moiety is a linear PEG. In some embodiments, the PEG moiety is a branched PEG.
  • the cell-penetrating peptides described herein are complexed with the one or more cargo molecules (e.g., a guide RNA described herein, a DNA nuclease described herein).
  • the cell-penetrating peptides are non-covalently complexed with at least one of the one or more cargo molecules.
  • the cell-penetrating peptides are non-covalently complexed with each of the one or more cargo molecules.
  • the cell-penetrating peptides are covalently complexed with at least one of the one or more cargo molecule.
  • the cell-penetrating peptides are covalently complexed with each of the one or more cargo molecules.
  • the complex or nanoparticles described herein comprise a guide RNA as described above.
  • the complex or nanoparticle comprises one or more genome-editing molecules (such as a DNA nuclease or a polynucleotide encoding the DNA nuclease).
  • the complex or nanoparticle described herein comprises a guide RNA that targets a mutated KRAS, such as any of the guide RNA described in the “synthetic guide RNAs” section.
  • the mutated KRAS comprises one or more mutations selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, GBR, G13A, G13D, G13V, GBP, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A146P, A146T and A146V.
  • the mutated KRAS comprises one or more mutations selected from the group consisting of G12D, G12C, G12V, G12A, G12S, G12R, G13D and G13C.
  • the guide RNA for targeting mutated KRAS described herein comprises a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, and 273-341.
  • crRNA specificity-determining CRISPR RNA
  • the nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19- 21, 23, 29, 31, 33, and 34.
  • the guide RNA is a single-guide RNA (sgRNA). In some embodiments, the guide RNA is 100% complementary to the target sequence and has the same length as the target sequence.
  • RNA e.g., a non-naturally occurring polynucleotide
  • a guide RNA for targeting mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 272-341.
  • the guide RNA is a singleguide RNA (sgRNA).
  • the guide RNA is 100% complementary to the target sequence and has the same length as the target sequence.
  • the present application further provides a composition, a complex (e.g., any of the complexes described herein), a nanoparticle, or a pharmaceutical formulation comprising any of the guide RNAs described above.
  • a composition, complex, nanoparticle, or pharmaceutical formulation described herein comprising any one or more (e.g., two, three, four or more) of these guide RNAs.
  • the nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34.
  • the guide RNA is a single-guide RNA (sgRNA).
  • the guide RNA is 100% complementary to the target sequence and has the same length as the target sequence.
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12V mutation
  • the guide RNA comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1-14 and 241-246.
  • the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8.
  • the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, and 8.
  • the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, and 8.
  • the target sequence is set forth in SEQ ID NO: 3.
  • the guide RNA comprises a nucleotide sequence 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, and 8. In some embodiments, the guide RNA comprises a nucleotide sequence 100% complementary to a target sequence of SEQ ID NO: 3.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 241.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 2.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 3.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 242.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 243.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 244.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 245.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 8.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 246.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 10.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 11.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS G12V mutation was present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12D mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 15-28 and 247-252.
  • the target sequence is selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23.
  • the target sequence is selected from the group consisting of SEQ ID NOs: 16, 19-21, and 23.
  • the target sequence is selected from the group consisting of SEQ ID NO: 19.
  • the guide RNA comprises a nucleotide sequence 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23. In some embodiments, the guide RNA comprises a nucleotide sequence 100% complementary to a target sequence of SEQ ID NO: 19.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12D, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 19.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 15.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 247.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 248.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12V, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 249.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12D, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 250.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12D, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 21.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 251.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 252.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12D, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 24.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 25.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at position 41-43.
  • the guide sequence has a length of about 20-24 base pairs, 20-22 base pairs, or 20-21 base pairs.
  • KRAS G12D mutation was present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12C mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 29-37, 253-257, 271, and 273.
  • the target sequence is selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34.
  • the target sequence is set forth in SEQ ID NO: 34.
  • the guide RNA comprises a nucleotide sequence 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34. In some embodiments, the guide RNA comprises a nucleotide sequence 100% complementary to a target sequence of SEQ ID NO: 34.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 34.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 29.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 253.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 254.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 255.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 33.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 33.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 34.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 256.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 257.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 273.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 271.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS G12C mutation was present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • Guide RNA targeting G12R can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12R mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 274-283.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 274.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 275.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 276.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 277.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 278.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 279.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 280.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 281.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 282.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 283.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS G12R is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12A mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 284-294.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 284.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 285.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 286.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 287.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 288.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 289.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 290.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 291.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 292.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 293.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 294.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS G12A is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12S mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 295-304.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 295.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 296.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 297.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 298.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 299.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 300.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 301.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 302.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 303.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 304.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS G12S is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G13D mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 305-309.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 305.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 306.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 307.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 308.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 309.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS G13D is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G13C mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 310-315.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 310.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 311.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 312.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 313.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 314.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 315.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS G13C is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a Q61H mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 316-322.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 316.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 317.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 318.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 319.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 320.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 321.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 322.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS Q61H is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • Guide RNA targeting Q61L can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a Q61L mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 323-329.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 323.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 324.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 325.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 326.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 327.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 328.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 329.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS Q61L is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • Guide RNA targeting A18D can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a A18D mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 330-332.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A18D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of CAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 330.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A18D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of CAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 331.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A18D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TTG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 332.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS A18D is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a KI 17N mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 333-335.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising KI 17N, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GTGA at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 333.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising KI 17N, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 334.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising KI 17N, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GTG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 333.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising KI 17N, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of CCT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 335.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS KI 17N is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a A146T mutation
  • the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 336-341.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 336.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 337.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 338.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of AAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 339.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TCA at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 340.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GTT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 341.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of CCT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 335.
  • the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.
  • KRAS A146T is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome).
  • exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.
  • the cancer is a malignant or advanced cancer.
  • Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).
  • the guide RNA is in the form of RNA.
  • the guide RNA is in the form of DNA encoding the RNA (i.e., gDNA).
  • the DNA is a plasmid DNA.
  • the plasmid DNA further comprises a DNA encoding a DNA nuclease (such as Cas9).
  • the guide RNA further comprises a DNA nuclease recruiting sequence.
  • the guide RNA is a single guide RNA (sgRNA) further comprising an auxiliary trans-activating crRNA (tracrRNA).
  • sgRNA single guide RNA
  • tracrRNA auxiliary trans-activating crRNA
  • the guide RNA further comprises a tracr mate sequence, a tracr sequence, and/or a tail sequence.
  • a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence.
  • degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence.
  • the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the guide sequence, tracr sequence and tracr mate sequence are contained within a single RNA (referred to herein as a “single-guide RNA,” or “sgRNA”), such that hybridization between the tracr sequence and the tracr mate sequence produces a secondary structure, such as a hairpin.
  • sgRNA single-guide RNA
  • Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences.
  • the sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG.
  • the sgRNA has at least two or more hairpins. In some embodiments, the sgRNA has two, three, four or five hairpins. In some embodiments, the sgRNA has at most five hairpins. In some embodiments, the sgRNA further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides.
  • the guide RNA is a prime editing guide RNA (pegRNA) that further comprises a primer binding sequence and/or a desired RNA sequence (for example at the 3 ’end of the guide RNA).
  • PegRNA can form a complex with a prime editor (such as a fusion protein comprising a modified Cas9 protein and a reverse transcriptase), thereby allowing prime editing of targeted sequences. See for example, Anzalone & Liu et al., Nature. 2019 Dec;576 (7785): 149- 157.
  • the guide RNA comprises one or more modification (e.g., chemical modification). .
  • the gRNA has one or more modified nucleotides, including nucleobase modification and/or backbone modification.
  • Exemplary modifications to the guide RNA include, but are not limited to, phosphorothioate backbone modification, 2’ -substitutions in the ribose (such as 2’-O-methyl and 2’-fluoro substitutions), LNA, and L-RNA.
  • the guide RNA does not have modifications to the nucleobase or backbone.
  • the guide RNA comprises a moiety that promotes the annealing of guide sequence.
  • the moiety comprises a synthetic nucleotide sequence, wherein the synthetic sequence is about 1-200 nucleotides, such as about 5 to about 100 nucleotides, such as about 8 to about 80 nucleotides, such as about 10 to about 50 nucleotides, such as about 12 to about 40 nucleotides.
  • the guide RNA (such as a single-guide RNA) has a length of no more than about 200 nucleotides, such as about 5 to about 100 nucleotides, such as about 8 to about 80 nucleotides, such as about 10 to about 50 nucleotides, such as about 12 to about 40 nucleotides.
  • RNAs can be employed to deliver any of the genome-editing complexes guide RNAs, complexes, nanoparticles, and compositions described in this application, including but not limited to, viral, liposome, electroporation, microinjection and conjugation, to achieve the introduction of the gRNA into a host cell.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding gRNA of the present invention to cells in culture, or in a host organism.
  • Non-viral vector delivery systems include DNA plasmids, RNA (e.g.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes for delivery to the host cell.
  • Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, electroporation, nanoparticles, exosomes, microvesicles, or genegun, naked DNA and artificial virions.
  • RNA or DNA viral based systems for the delivery of nucleic acids has high efficiency in targeting a virus to specific cells and trafficking the viral payload to the cellular nuclei.
  • the complex or nanoparticle described herein further comprises a DNA nuclease or a nucleotide encoding the DNA nuclease.
  • the DNA nuclease is selected from the group consisting of a CRISPR- associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof.
  • Cas CRISPR- associated protein
  • ZFN zinc finger nuclease
  • TALEN transcription activatorlike effector nuclease
  • a complex or nanoparticle described herein comprises an RGEN (e.g., Cas9).
  • the protein or polypeptide is between about 10 kDa and about 200 kDa (such as about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 kDa, including any ranges between these values).
  • the complex or nanoparticle comprises a plurality of proteins or polypeptides, wherein each of the plurality of protein or polypeptides is between about 10 kDa and about 200 kDa (such as about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 kDa, including any ranges between these values).
  • a complex or nanoparticle described herein further comprises a nucleic acid encoding a DNA nuclease.
  • the nucleic acid is between about 20 nt and about 20 kb (such as about any of 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 kb, including any ranges between these values).
  • the nucleic acid is DNA, such as a DNA plasmid encoding a genome-editing system molecule.
  • the DNA plasmid comprises an expression cassette for expressing the genome-editing system molecule.
  • the DNA plasmid is between about 1 kb and about 20 kb (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 kb, including any ranges between these values).
  • the nucleic acid is RNA, such as mRNA encoding a genome-editing system molecule.
  • the mRNA is between about 100 nt and about 10 kb (such as about any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 kb, including any ranges between these values).
  • the complex or nanoparticle comprises a plurality of nucleic acids, such as any of the nucleic acids described herein.
  • the complex or nanoparticle comprises a gRNA and a nucleic acid encoding a genomeediting system molecule (e.g., a DNA plasmid or mRNA encoding the DNA nuclease).
  • the complex or nanoparticle comprises nucleic acid encoding a plurality of genome-editing system molecules (e.g., one or more DNA plasmid encoding the plurality of genome-editing system molecules, or a plurality of mRNAs encoding the plurality of genome-editing system molecules).
  • the nucleic acids are single stranded oligonucleotides. In some embodiments, the nucleic acids are double stranded oligonucleotides.
  • the nucleic acids described herein may be any of a range of length of up to, but not necessarily 200 nucleotides in the case of antisense oligonucleotides, RNAi, siRNA, shRNA, iRNA, antagomirs or up to 1000 kilo bases in the case of plasmid DNA.
  • the nucleic acids are plasmid DNA or DNA fragments (for example DNA fragments of lengths of up to about 1000 bp).
  • the plasmid DNA or DNA fragments may be hypermethylated or hypomethylated.
  • the plasmid DNA or DNA fragments encode one or more genes, and may contain regulatory elements necessary for the expression of said one or more genes.
  • the plasmid DNA or DNA fragments may comprise one or more genes that encode a selectable marker, allowing for maintenance of the plasmid DNA or DNA fragment in an appropriate host cell.
  • the DNA nuclease is a CRISPR-associated nuclease.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • SPIDRs Sacer Interspersed Direct Repeats
  • the CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associated genes.
  • SSRs interspersed short sequence repeats
  • the CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).
  • SRSRs short regularly spaced repeats
  • the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., [2000], supra).
  • the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401
  • CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 [2002]; and Mojica et al., [2005]) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter
  • Acinetobacter Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.
  • the DNA nuclease comprises a CRIS PR-associated nuclease, e.g., a Cas protein that is an RNA guided nuclease.
  • the Cas protein is a nuclease that binds to a guide RNA which recognizes a target sequence.
  • the Cas protein may a single or double strand into the target site.
  • CRISPR-Cas systems fall into two major classes: class 1 systems use a complex of multiple Cas proteins to degrade nucleic acids; class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV; and class 2 is divided into types II, V, and VI.
  • Cas proteins adapted for gene editing applications include, but are not limited to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Casio, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, and Mad7.
  • Cas9 is a type II Cas protein and is described herein as illustrative. These Cas proteins may be originated from different source species. For example, Cas9 can be derived from S. pyogenes or S. aureus.
  • the type II CRISPR system incorporates sequences from invading DNA between CRISPR repeat sequences encoded as arrays within the host genome. Transcripts from the CRISPR repeat arrays are processed into CRISPR RNAs (crRNAs), each harboring a variable sequence transcribed from the invading DNA, known as the “protospacer” sequence, as well as part of the CRISPR repeat. Each crRNA hybridizes with a second transactivating CRISPR RNA (tracrRNA), and these two RNAs form a complex with the Cas9 nuclease. The protospacer-encoded portion of the crRNA directs the Cas9 complex to cleave complementary target DNA sequences, provided that they are adjacent to short sequences known as “protospacer adjacent motifs” (PAMs).
  • PAMs protospacer adjacent motifs
  • Cpfl CRISPR from Prevotella and Franciscella 1; also known as Casl2a
  • Casl2a is an RNA-guided nuclease that only requires a crRNA and does not need a tracrRNA to function.
  • synthetic gRNAs gRNAs can be single guide RNAs (sgRNAs such as any of those described above) composed of a crRNA, a tetraloop, and a tracrRNA.
  • the crRNA usually comprises a complementary region (also called a spacer, usually about 20 nucleotides in length) that is user-designed to recognize a target DNA of interest.
  • the tracrRNA sequence comprises a scaffold region for Cas nuclease binding.
  • the crRNA sequence and the tracrRNA sequence are linked by the tetraloop and each have a short repeat sequence for hybridization with each other, thus generating a chimeric sgRNA.
  • One can change the genomic target of the Cas nuclease by simply changing the spacer or complementary region sequence present in the gRNA.
  • the complementary region will direct the Cas nuclease to the target DNA site through standard RNA-DNA complementary base pairing rules.
  • Cas nucleases may comprise one or more mutations to alter their activity, specificity, recognition, and/or other characteristics.
  • the Cas nuclease may have one or more mutations that alter its fidelity to mitigate off-target effects (e.g., eSpCas9, SpCas9-HFl, FlypaSpCas9, FleFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9).
  • the Cas nuclease has one or more inactive nuclease domains.
  • the genome editing enzyme is an RNA guided nuclease.
  • the RNA guided nuclease is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas 10, Cas 12, Cas 12a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, C
  • the DNA nuclease is a Cas protein.
  • Cas proteins include Casl, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, such as inducible
  • pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2, and the amino acid sequence of Acidaminococcus sp. Cpfl protein may be found in the SwissProt database under accession number U2UMQ6.
  • the DNA nuclease comprises an unmodified or modified CRISPR enzyme that has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae.
  • the CRISPR enzyme is Cpfl, and may be Cpfl from Acidaminococcus or Lachnospiraceae.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • the CRISPR enzyme is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • D10A aspartate-to-alanine substitution
  • pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • a Cas9 nickase may be used in combination with guide sequences, e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ.
  • two or more catalytic domains of Cas9 may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity.
  • a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.
  • a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form.
  • Other mutations may be useful; where the Cas9 or other CRISPR enzyme is from a species other than S. pyogenes, mutations in corresponding amino acids may be made to achieve similar effects.
  • the Cas protein (such as Cas9) is a split Cas protein comprising an N-terminal Cas protein fragment, Cas(N), and a C-terminal Cas protein fragment, Cas(C), wherein Cas(N) is fused to a first dimerization domain and Cas(C) is fused to a second dimerization domain, and wherein the first and second dimerization domains facilitate dimerization of Cas(N) and Cas(C) to form a complex with a functional Cas nuclease activity.
  • dimerization of the first and second dimerization domains is sensitive to a dimerization agent.
  • the first and second dimerization domains comprise the FK506 binding protein 12 (FKBP) and FKBP rapamycin binding (FRB) domains of the mammalian target of rapamycin (mTOR), and the dimerization agent is rapamycin.
  • FKBP FK506 binding protein 12
  • FKBP rapamycin binding domains of the mammalian target of rapamycin (mTOR)
  • mTOR mammalian target of rapamycin
  • the complex or nanoparticle described herein comprises a nucleotide sequence encoding a CRISPR enzyme (such as Cas9 endonuclease) is codon optimized for expression in particular cells, such as eukaryotic cells.
  • a CRISPR enzyme such as Cas9 endonuclease
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g.
  • Codon bias differences in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available.
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • the CRISPR enzyme described herein comprises one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • NLSs nuclear localization sequences
  • the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus).
  • the CRISPR enzyme comprises at most 6 NLSs.
  • an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known.
  • Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 212); the NLS from nucleoplasmin (e.g.
  • the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 213)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 214) or RQRRNELKRSP (SEQ ID NO: 215); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 216); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 217) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 218) and PPKKARED (SEQ ID NO: 219) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 220) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 221) of mouse c-abl IV;
  • the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme).
  • a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • Nonlimiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta- glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-gal
  • a CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.
  • MBP maltose binding protein
  • DBD Lex A DNA binding domain
  • HSV herpes simplex virus
  • the cargo comprises a base editor.
  • Base editors have been developed that convert Cas endonucleases into programmable nucleotide deaminases, thus facilitating the introduction of C-to-T mutations (by C-to-U deamination) or A-to-G mutations (by A-to-I deamination) without induction of a double-strand break.
  • Base editors comprise a nickase form of SpCas9 (nSpCas9, to stimulate cellular DNA mismatch repair) fused to a nucleobase deaminase enzyme as well as an inhibitor of base excision repair such as uracil glycosylase inhibitor (UGI).
  • the cargo comprises a prime editor.
  • Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and provide the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. See Anzalone et al. Search-and-replace genome editing without doublestrand breaks or donor DNA. Nature volume 576, pages 149-157(2019).
  • pegRNA prime editing guide RNA
  • the cargo comprises a fusion protein comprising a catalytically disabled nuclease (such as a catalytically disabled Cas9 endonuclease) and a reversed transcriptase (such as a pentamutant of M-MLV reverse transcriptase).
  • a catalytically disabled nuclease such as a catalytically disabled Cas9 endonuclease
  • a reversed transcriptase such as a pentamutant of M-MLV reverse transcriptase.
  • the cargo comprises a polynucleotide encoding the fusion protein.
  • the cargo comprises a fusion protein comprising a catalytically disabled nuclease (such as a catalytically disabled Cas9 endonuclease) and a nucleobase deaminase enzyme.
  • the nucleobase deaminase enzyme is APOBEC1 cytidine deaminase.
  • the nucleobase deaminase enzyme is cytidine deaminase CDA1.
  • the fusion protein further comprises a DNA glycosylase inhibitor.
  • the DNA glycosylase inhibitor is uracil DNA glycosylase inhibitor (UGI).
  • the cargo comprises a polynucleotide encoding the fusion protein.
  • ZFPs and ZFNs ZFPs and ZFNs; TALs, TALEs, and TALENs
  • the cargo molecule includes a DNA-binding protein such as one or more zinc finger protein (ZFP) or transcription activator-like protein (TAL), fused to an effector protein such as an endonuclease (or nucleic acid encoding the DNA-binding protein/effector protein fusion).
  • ZFP zinc finger protein
  • TAL transcription activator-like protein
  • an effector protein such as an endonuclease (or nucleic acid encoding the DNA-binding protein/effector protein fusion).
  • ZFNs zinc finger protein
  • TALEs transcription activator-like protein
  • TALENs or nucleic acid encoding the DNA-binding protein/effector protein fusion.
  • the guide RNA described herein can be in the form of a DNA (i.e., guide DNA, gDNA) encoding the RNA that guides ZFP or TAL to the target set.
  • the cargo molecule comprises one or more zinc-finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner.
  • ZFPs zinc-finger proteins
  • a ZFP or domain thereof is a protein or domain within a larger protein that binds DNA in a sequencespecific manner through one or more zinc fingers, regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
  • ZFPs are artificial ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers.
  • ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers.
  • sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3 and 6) on a zinc finger recognition helix.
  • the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is engineered to bind to a target site of choice. See, for example, Beerli et al. (2002) Nature Biotechnol.
  • the cargo molecule includes a zinc-finger DNA binding domain fused to a DNA cleavage domain to form a zinc-finger nuclease (ZFN).
  • fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
  • the cleavage domain is from the Type IIS restriction endonuclease Fok I. Fok I generally catalyzes double- stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other.
  • ZFNs target a gene present in a target cell.
  • the ZFNs efficiently generate a double strand break (DSB), for example at a predetermined site in the coding region of the gene.
  • Typical regions targeted include exons, regions encoding N- terminal regions, first exon, second exon, and promoter or enhancer regions.
  • transient expression of the ZFNs promotes highly efficient and permanent disruption of the target gene in target cells.
  • delivery of the ZFNs results in the permanent disruption of the gene with efficiencies surpassing 50%.
  • the cargo molecule includes a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference in its entirety herein.
  • TAL transcription activator-like protein
  • TALE transcription activator-like protein effector
  • a TALE DNA binding domain or TALE is a polypeptide comprising one or more TALE repeat domains/units.
  • the repeat domains are involved in binding of the TALE to its cognate target DNA sequence.
  • a single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein.
  • Each TALE repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Diresidue (RVD), typically at positions 12 and/or 13 of the repeat.
  • RVD Repeat Variable Diresidue
  • TALEs The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NG binds to T and non-canonical (atypical) RVDs are also known. See, U.S. Patent Publication No. 20110301073.
  • TALEs may be targeted to any gene by design of TAL arrays with specificity to the target DNA sequence.
  • the target sequence generally begins with a thymidine.
  • the cargo molecule includes a DNA binding endonuclease, such as a TALE-nuclease (TALEN).
  • TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a nuclease catalytic domain to cleave a nucleic acid target sequence.
  • the TALE DNA-binding domain has been engineered to bind a target sequence within a target gene.
  • the TALEN recognizes and cleaves the target sequence in the gene.
  • cleavage of the DNA results in double- stranded breaks.
  • the breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • repair mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson, Trends Biochem Sci. 1998 Oct;23(10):394-8) or via the so-called microhomology mediated end joining.
  • repair via NHEJ results in small insertions or deletions and can be used to disrupt and thereby repress the gene.
  • the modification may be a substitution, deletion, or addition of at least one nucleotide.
  • cells in which a cleavage-induced mutagenesis event, i.e. a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and/or selected by well-known methods in the art.
  • TALE repeats are assembled to specifically target a gene.
  • a library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., Nature Biotechnology . 31, 251-258 (2013)).
  • Custom-designed TALE arrays are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA).
  • the TALENs are introduced as transgenes encoded by one or more plasmid vectors.
  • the plasmid vector can contain a selection marker which provides for identification and/or selection of cells which received said vector.
  • the DNA nuclease comprises a meganuclease.
  • Meganucleases are enzymes in the endonuclease family that are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). Meganucleases are grouped into families based on their structural motifs, which affect nuclease activity and/or DNA recognition. The most widespread and best known meganucleases are the proteins in the LAGLID ADG family, which owe their name to a conserved amino acid sequence. See Chevalier et al., Nucleic Acids Res. (2001) 29(18): 3757-3774.
  • the GIY- YIG family members have a GIY-YIG module, which is 70-100 residues long and includes four or five conserved sequence motifs with four invariant residues, two of which are required for activity. See Van Roey et al., Nature Struct. Biol. (2002) 9:806-811.
  • the His-Cys family meganucleases are characterized by a highly conserved series of histidines and cysteines over a region encompassing several hundred amino acid residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.
  • NHN family are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774. [0410] Because the chance of identifying a natural meganuclease for a particular target DNA sequence is low due to the high specificity requirement, various methods including mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Strategies for engineering a meganuclease with altered DNA-binding specificity, e.g., to bind to a predetermined nucleic acid sequence are known in the art.
  • Meganucleases can create DSBs in the genomic DNA, which can create a frame-shift mutation if improperly repaired, e.g., via NHEJ, leading to a decrease in the expression of a target gene in a cell.
  • foreign DNA can be introduced into the cell along with the meganuclease. Depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify the target gene. See Silva et al., Current Gene Therapy (2011) 11 :11 -27.
  • the DNA nuclease comprises a transposase.
  • Transposases are enzymes that bind to the end of a transposon and catalyze its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism.
  • transposases By linking transposases to other systems such as the CRISPR/Cas system, new gene editing tools can be developed to enable site specific insertions or manipulations of the genomic DNA.
  • transposons which use a catalytically inactive Cas effector protein and Tn7-like transposons. The transposase-dependent DNA integration does not provoke DSBs in the genome, which may guarantee safer and more specific DNA integration.
  • the complex or nanoparticles described herein further comprises a donor nucleic acid.
  • the donor nucleic acid is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex.
  • a donor nucleic acid may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length.
  • the donor nucleic acid comprises a sequence that is complementary to a portion of a polynucleotide comprising the target sequence.
  • the donor nucleic acid overlaps with one or more nucleotides of the target sequence (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides).
  • the nearest nucleotide of the donor nucleic acid in the region of complementarity is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
  • the cargo comprises one or more RNAi e.g., siRNA) that targets a mutant form of KRAS.
  • the mutant form of KRAS comprises an aberration of KRAS, wherein the aberration of KRAS comprises a mutation on codon 12, 13, 17, 18, 34, 59, 61, 68, 95, 96, 117, and/or 146 of KRAS.
  • the aberration of KRAS comprises a mutation on codon 12, or 61 of KRAS.
  • the aberration of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, GBR, G13A, G13D, G13V, GBP, S17G, A18D, P34S, A59T, Q61H, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, R68M, R68S, H95D, H95Q, H95R, Y96C, Y96D, K117N, A146P, A146T and A146V.
  • the aberration of KRAS is selected from the group consisting of G12C, G12S, GBR, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, GBP, S17G, P34S, Q61K, Q61L, Q61R, and Q61H.
  • the aberration of KRAS is selected from the group consisting of G12C, GBR, G12S, G12A, G12D, G12V, G13C, GBR, G13S, G13A, G13D, G13V, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T and A146V.
  • the aberration of KRAS is selected from the group consisting of KRAS G12A, G12C, G12D, GBR, G12S, G12V, G13A, G13C, G13D, GBR, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, and Q61R.
  • the aberration of KRAS is selected from the group consisting of KRAS G12C, G12D, GBR, G12S, G12V and G13D. In some embodiments, the aberration of KRAS is selected from G12C, G12D and Q61K. [0415] In some embodiments, the one or more RNAi (e.g., siRNA) is selected from the group consisting of SEQ ID NOs: 228-234.
  • siRNA e.g., siRNA
  • a complex or nanoparticle as described herein comprises a targeting moiety, wherein the targeting moiety is a ligand capable of cell-specific and/or nuclear targeting.
  • a cell membrane surface receptor and/or cell surface marker is a molecule or structure which can bind said ligand with high affinity and preferably with high specificity. Said cell membrane surface receptor and/or cell surface marker is preferably specific for a particular cell, i.e. it is found predominantly in one type of cell rather than in another type of cell (e.g. galactosyl residues to target the asialoglycoprotein receptor on the surface of hepatocytes).
  • the cell membrane surface receptor facilitates cell targeting and internalization into the target cell of the ligand (e.g.
  • ligand moieties/ligand binding partners that may be used in the context of the present application are widely described in the literature.
  • Such a ligand moiety is capable of conferring to the complex or nanoparticle of the application the ability to bind to a given binding-partner molecule or a class of binding-partner molecules localized at the surface of at least one target cell.
  • Suitable binding-partner molecules include without limitation polypeptides selected from the group consisting of cell-specific markers, tissue-specific markers, cellular receptors, viral antigens, antigenic epitopes and tumor-associated markers.
  • Binding-partner molecules may moreover consist of or comprise, for example, one or more sugar, lipid, glycolipid, antibody molecules or fragments thereof, or aptamer.
  • a ligand moiety may be for example a lipid, a glycolipid, a hormone, a sugar, a polymer (e.g. PEG, polylysine, PET), an oligonucleotide, a vitamin, an antigen, all or part of a lectin, all or part of a polypeptide, such as for example JTS1 (WO 94/40958), an antibody or a fragment thereof, or a combination thereof.
  • the ligand moiety used in the present application is a peptide or polypeptide having a minimal length of 7 amino acids. It is either a native polypeptide or a polypeptide derived from a native polypeptide. “Derived” means containing (a) one or more modifications with respect to the native sequence (e.g. addition, deletion and/or substitution of one or more residues), (b) amino acid analogs, including non-naturally occurring amino acids, (c) substituted linkages, or (d) other modifications known in the art.
  • polypeptides serving as ligand moiety encompass variant and chimeric polypeptides obtained by fusing sequences of various origins, such as for example a humanized antibody which combines the variable region of a mouse antibody and the constant region of a human immunoglobulin.
  • polypeptides may have a linear or cyclized structure (e.g. by flanking at both extremities a polypeptide ligand by cysteine residues).
  • the polypeptide in use as a ligand moiety may include modifications of its original structure by way of substitution or addition of chemical moieties (e.g. glycosylation, alkylation, acetylation, amidation, phosphorylation, addition of sulfhydryl groups and the like).
  • the application further contemplates modifications that render the ligand moiety detectable.
  • modifications with a detectable moiety can be envisaged (i.e. a scintigraphic, radioactive, or fluorescent moiety, or a dye label and the like).
  • detectable labels may be attached to the ligand moiety by any conventional techniques and may be used for diagnostic purposes (e.g. imaging of tumoral cells).
  • the binding-partner molecule is an antigen (e.g. a target cell-specific antigen, a disease-specific antigen, an antigen specifically expressed on the surface of engineered target cells) and the ligand moiety is an antibody, a fragment or a minimal recognition unit thereof (e.g.
  • the ligand moiety may be a monoclonal antibody. Many monoclonal antibodies that bind many of these antigens are already known, and using techniques known in the art in relation to monoclonal antibody technology, antibodies to most antigens may be prepared.
  • the ligand moiety may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example, ScFv). In some embodiments, the ligand moiety is selected among antibody fragments, rather than whole antibodies. Effective functions of whole antibodies, such as complement binding, are removed.
  • ScFv and dAb antibody fragments may be expressed as a fusion with one or more other polypeptides.
  • Minimal recognition units may be derived from the sequence of one or more of the complementary-determining regions (CDR) of the Fv fragment.
  • Whole antibodies, and F(ab')2 fragments are “bivalent”. By “bivalent” it is meant that said antibodies and F(ab')2 fragments have two antigen binding sites.
  • Fab, Fv, ScFv, dAb fragments and minimal recognition units are monovalent, having only one antigen binding sites.
  • the ligand moiety allows targeting to a tumor cell and is capable of recognizing and binding to a molecule related to the tumor status, such as a tumor- specific antigen, a cellular protein differentially or over-expressed in tumor cells or a gene product of a cancer-associated vims.
  • a tumor-specific antigen include but are not limited to MUC-1 related to breast cancer (Hareuven i el al., 990, Eur. J.
  • Biochem 189, 475-486 the products encoded by the mutated BRCA1 and BRCA2 genes related to breast and ovarian cancers (Miki et al, 1994, Science 226, 66-7 1; Fuireal et al, 1994, Science 226, 120- 122; Wooster et al., 1995, Nature 378, 789-792), APC related to colon cancer (Poiakis, 1995, Curr. Opin. Genet. Dev. 5, 66-71), prostate specific antigen (PSA) related to prostate cancer, (Stamey et al., 1987, New England J. Med. 317, 909), carcinoma embryonic antigen (CEA) related to colon cancers (Schrewe et al., 1990, Mol. Cell.
  • PSA prostate specific antigen
  • CEA carcinoma embryonic antigen
  • the ligand moiety is a fragment of an antibody capable of recognizing and binding to the MUC-1 antigen and thus targeting MUC-1 positive tumor cells.
  • the ligand moiety is the scFv fragment of the SM3 monoclonal antibody which recognizes the tandem repeat region of the MUC- 1 antigen (Burshell et al. , 1987, Cancer Res. 47, 5476-5482; Girling et al., 1989, Int. J. Cancer 43, 1072-1076; Dokumo et al., 1998, J. Mol. Biol. 284, 713-728).
  • Examples of cellular proteins differentially or overexpressed in tumor cells include but are not limited to the receptor for interleukin 2 (IL- 2) overexpressed in some lymphoid tumors, GRP (Gastrin Release Peptide) overexpressed in lung carcinoma cells, pancreas, prostate and stomach tumors (Michael et al., 1995, Gene Therapy 2, 660-668), TNF (Tumor Necrosis Factor) receptor, epidermal growth factor receptors, Fas receptor, CD40 receptor, CD30 receptor, CD27 receptor, OX-40, a-v integrins (Brooks et al, 994, Science 264, 569) and receptors for certain angiogenic growth factors (Hanahan, 1997, Science 277, 48).
  • IL-2 interleukin 2
  • GRP Gastrin Release Peptide
  • TNF Tumor Necrosis Factor
  • epidermal growth factor receptors Fas receptor
  • CD40 receptor CD30 receptor
  • CD27 receptor CD27 receptor
  • OX-40
  • IL-2 is a suitable ligand moiety to bind to TL-2 receptor.
  • receptors that are specific to fibrosis and inflammation, these include the TGFbeta receptors or the Adenosine receptors that are identified above and are suitable targets for application compositions.
  • Cell surface markers for multiple myeloma include, but are not limited to, CD56, CD40, FGFR3, CS1, CD138, IGF1R, VEGFR, and CD38, and are suitable targets for application compositions.
  • Suitable ligand moieties that bind to these cell surface markers include, but are not limited to, anti-CD56, anti-CD40, PRO-001, Chir-258, HuLuc63, anti-CD138-DMl, anti-IGFIR and bevacizumab.
  • a complex or nanoparticle described herein comprises one or more molecules of a genome-editing system (such as the entire genome-editing system) targeting one or more genes including, but are not limited to, Adenosine receptor A2A, Adenosine receptor A2B, Adenylyl cyclase, Akt, ALK, ALK/Met, angiopoietin receptor, Angiotensin II, APC, AR, ARK5, arrestin, ATF1, ATF-2, B7-1, B7-hl (pdl-1), P-catenin, Bcl-2, BCL2L12, Bcl6, Bcr-Abl, BRAF, BRCA1, BRCA2, BTK, caspase-2, caspase-9, CCL2, CCN1, Ccnd2, CDK-activating kinases, CEB PA, Chop, c-Jun, c-Myc, CREB, CREB1, CS1, CTGF, CTNNB1,
  • the complex or nanoparticle comprises one or more molecules of an RGEN-based genomeediting system (e.g., a CRISPR/Cas9 genome-editing system), wherein the RGEN-based genome-editing system comprises a gRNA targeting one of the genes described herein.
  • the complex or nanoparticle comprises one or more molecules of a ZFN- based genome-editing system, wherein the ZFN targets one of the genes described herein.
  • the complex or nanoparticle comprises one or more molecules of a TALEN-based genome-editing system, wherein the TALEN targets one of the genes described herein.
  • the complex or nanoparticle comprises one or more molecules of a homing endonuclease-based genome-editing system, wherein the homing endonuclease targets one of the genes described herein. In some embodiments, the complex or nanoparticle comprises one or more molecules of an integrase-based genome-editing system, wherein the integrase targets one of the genes described herein.
  • the genome-editing complexes described herein is in a nanoparticle comprising a core comprising the genome-editing complexes described above.
  • the nanoparticle comprises a core comprising a complex described herein, wherein the cell-penetrating peptide in the genome-editing delivery complex is associated with the cargo.
  • the association is non-covalent. In some embodiments, the association is covalent.
  • the nanoparticle further comprises a surface layer (e.g., a shell) comprising a peripheral cell-penetrating peptide (z.e., CPP), wherein the core is coated by the shell.
  • a surface layer e.g., a shell
  • a peripheral cell-penetrating peptide z.e., CPP
  • the core is coated by the shell.
  • the peripheral CPP is the same as a CPP in the core.
  • the peripheral CPP is different than any of the CPPs in the core.
  • the peripheral CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a poly-arginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (such as a VEPEP-3, VEPEP-4, VEPEP-5, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide.
  • the peripheral CPP is a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN- 100 peptide.
  • the peripheral cell-penetrating peptide is selected from the group consisting of PEP- 1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • at least some of the peripheral cell-penetrating peptides in the surface layer are linked to a targeting moiety.
  • the linkage is covalent.
  • the covalent linkage is by chemical coupling.
  • the covalent linkage is by genetic methods.
  • the nanoparticle further comprises an intermediate layer between the core of the nanoparticle and the surface layer.
  • the intermediate layer comprises an intermediate CPP.
  • the intermediate CPP is the same as a CPP in the core.
  • the intermediate CPP is different than any of the CPPs in the core.
  • the intermediate CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a poly-arginine- based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (such as a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide.
  • the intermediate CPP is a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide.
  • the nanoparticle comprises two or more guide RNAs such as any one of the guide RNAs described herein.
  • the two or more guide RNAs targets two or more different KRAS mutations.
  • the two or more different KRAS mutations are selected from the group consisting of G12D, G12V, and G12C.
  • the two or more guide RNAs are contained in the same genomeediting complex. In some embodiments, the two or more guide RNAs are contained in different genome-editing complex.
  • the nanoparticle core comprises a plurality of genome-editing complexes. In some embodiments, the nanoparticle core comprises a plurality of genomeediting complexes present in a predetermined ratio. In some embodiments, the predetermined ratio is selected to allow the most effective use of the nanoparticle in any of the methods described below in more detail. In some embodiments, the nanoparticle core further comprises one or more additional guide RNAs, one or more additional cell-penetrating peptides, one or more additional genome-editing nucleases, and/or one or more additional donor nucleic acids.
  • the one or more additional complex comprises at least one or more the guide RNAs that targets a different KRAS mutation.
  • the nanoparticle described herein comprises a) a first complex comprising a first guide RNA that specifically targeting G12D (such as any one of the guide RNA targeting G12D described herein), and b) a second complex comprising a second guide RNA that specifically targeting G12V (such as any one of the guide RNA targeting G12V described herein).
  • the nanoparticle described herein comprises a) a first complex comprising a first guide RNA that specifically targeting G12D (such as any one of the guide RNA targeting G12D described herein), and b) a second complex comprising a second guide RNA that specifically targeting G12C (such as any one of the guide RNA targeting G12C described herein).
  • the nanoparticle described herein comprises a) a first complex comprising a first guide RNA that specifically targeting G12C (such as any one of the guide RNA targeting G12C described herein), and b) a second complex comprising a second guide RNA that specifically targeting G12V (such as any one of the guide RNA targeting G12V described herein).
  • the nanoparticle described herein comprises a) a first complex comprising a first guide RNA that specifically targeting G12D (such as any one of the guide RNA targeting G12D described herein), b) a second complex comprising a second guide RNA that specifically targeting G12V (such as any one of the guide RNA targeting G12V described herein), and c) a second complex comprising a second guide RNA that specifically targeting G12V (such as any one of the guide RNA targeting G12V described herein).
  • the nanoparticle further comprises one or more additional cellpenetrating peptides.
  • the one or more additional cell-penetrating peptides include, but are not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (such as a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide.
  • at least some of the one or more additional cellpenetrating peptides are linked to a targeting moiety. In some embodiments, the linkage is covalent.
  • the mean size (diameter) of the nanoparticle is from about 20 nm to about 1000 nm, including for example from about 50 nm to about 800 nm, from about 75 nm to about 600 nm, from about 100 nm to about 600 nm, and from about 200 nm to about 400 nm. In some embodiments, the mean size (diameter) of the nanoparticle is no greater than about 1000 nanometers (nm), such as no greater than about any of 900, 800, 700, 600, 500, 400, 300, 200, or 100 nm.
  • the average or mean diameter of the nanoparticle is no greater than about 200 nm. In some embodiments, the average or mean diameters of the nanoparticles is no greater than about 150 nm. In some embodiments, the average or mean diameter of the nanoparticle is no greater than about 100 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 20 nm to about 400 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 30 nm to about 400 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 40 nm to about 300 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 50 nm to about 200 nm.
  • the average or mean diameter of the nanoparticle is about 60 nm to about 150 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 70 nm to about 100 nm. In some embodiments, the nanoparticles are sterile-filterable.
  • the zeta potential of the nanoparticle is from about -30 mV to about 60 mV (such as about any of -30, -25, -20, -15, -10, -5, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 mV, including any ranges between these values).
  • the zeta potential of the nanoparticle is from about -30 mV to about 30 mV, including for example from about -25 mV to about 25 mV, from about -20 mV to about 20 mV, from about -15 mV to about 15 mV, from about -10 mV to about 10 mV, and from about -5 mV to about 10 mV.
  • the polydispersity index (PI) of the nanoparticle is from about 0.05 to about 0.6 (such as about any of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, and 0.6, including any ranges between these values).
  • the nanoparticle is substantially non-toxic.
  • the complex or the nanoparticle described herein is in a composition (e.g., a pharmaceutical composition) comprising a complex or nanoparticle as described herein.
  • the composition is a pharmaceutical composition comprising a complex or nanoparticle as described herein and a pharmaceutically acceptable diluent, excipient, and/or carrier.
  • the composition comprises a mixture of two or more nanoparticles, wherein the two or more nanoparticles comprise different guide RNAs that target different KRAS mutations.
  • the composition comprises a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12D, and b) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12V.
  • the composition comprises a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12D, and b) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12C.
  • the composition comprises a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12C, and b) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12V.
  • the composition comprises a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12D, b) a third nanoparticle comprising a second guide RNA that specifically targets KRAS G12V, c) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12C.
  • the concentration of the complex or nanoparticle in the composition is from about 1 nM to about 100 mM, including for example from about 10 nM to about 50 mM, from about 25 nM to about 25 mM, from about 50 nM to about 10 mM, from about 100 nM to about 1 mM, from about 500 nM to about 750 pM, from about 750 nM to about 500 pM, from about 1 pM to about 250 pM, from about 10 pM to about 200 pM, and from about 50 pM to about 150 pM.
  • the pharmaceutical composition is lyophilized.
  • diluent, excipient, and/or carrier as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other vertebrate hosts.
  • a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals.
  • diluent, excipient, and/or “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered.
  • Such pharmaceutical diluent, excipient, and/or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions.
  • Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like, including lyophilization aids.
  • the composition if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like.
  • a composition comprising a complex or nanoparticle as described herein further comprises a pharmaceutically acceptable diluent, excipient, and/or carrier.
  • the pharmaceutically acceptable diluent, excipient, and/or carrier affects the level of aggregation of a complex or nanoparticle in the composition and/or the efficiency of intracellular delivery mediated by a complex or nanoparticle in the composition.
  • the extent and/or direction of the effect on aggregation and/or delivery efficiency mediated by the pharmaceutically acceptable diluent, excipient, and/or carrier is dependent on the relative amount of the pharmaceutically acceptable diluent, excipient, and/or carrier in the composition.
  • the presence of a pharmaceutically acceptable diluent, excipient, and/or carrier does not promote and/or contribute to aggregation of the complex or nanoparticle, or promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the complex or nanoparticle.
  • a pharmaceutically acceptable diluent, excipient, and/or carrier such as a salt, sugar, chemical buffering agent, buffer solution, cell culture medium, or carrier protein
  • the composition comprises the pharmaceutically acceptable diluent, excipient, and/or carrier at a concentration that does not promote and/or contribute to aggregation of the complex or nanoparticle, or promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the complex or nanoparticle.
  • a concentration that does not promote and/or contribute to aggregation of the complex or nanoparticle, or promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60
  • the composition comprises the pharmaceutically acceptable diluent, excipient, and/or carrier at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 150% larger than the size of the complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and/or carrier at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 100% larger than the size of the complex or nanoparticle.
  • the composition comprises the pharmaceutically acceptable diluent, excipient, and/or carrier at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 50% larger than the size of the complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and/or carrier at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 20% larger than the size of the complex or nanoparticle.
  • the composition comprises the pharmaceutically acceptable diluent, excipient, and/or carrier at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 15% larger than the size of the complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and/or carrier at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 10% larger than the size of the complex or nanoparticle. In some embodiments, the pharmaceutically acceptable diluent, excipient, and/or carrier is a salt, including, without limitation, NaCl.
  • the pharmaceutically acceptable diluent, excipient, and/or carrier is a sugar, including, without limitation, sucrose, glucose, and mannitol.
  • the pharmaceutically acceptable diluent, excipient, and/or carrier is a chemical buffering agent, including, without limitation, HEPES.
  • the pharmaceutically acceptable diluent, excipient, and/or carrier is a buffer solution, including, without limitation, PBS.
  • the pharmaceutically acceptable diluent, excipient, and/or carrier is a cell culture medium, including, without limitation, DMEM.
  • Particle size can be determined using any means known in the art for measuring particle size, such as by dynamic light scattering (DLS). For example, in some embodiments, an aggregate having a Z-average as measured by DLS that is 10% greater than the Z-average as measured by DLS of a complex or nanoparticle is 10% larger than the complex or nanoparticle.
  • DLS dynamic light scattering
  • the composition comprises a salt (e.g., NaCl) at a concentration that does not promote and/or contribute to aggregation of the complex or nanoparticle, or promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 100% (such as no more than about any of 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the complex or nanoparticle.
  • a salt e.g., NaCl
  • the composition comprises a salt (e.g., NaCl) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 75% larger than the size of the complex or nanoparticle.
  • the composition comprises a salt (e.g., NaCl) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 50% larger than the size of the complex or nanoparticle.
  • the composition comprises a salt (e.g., NaCl) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 20% larger than the size of the complex or nanoparticle.
  • the composition comprises a salt (e.g., NaCl) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 15% larger than the size of the complex or nanoparticle.
  • the composition comprises a salt (e.g., NaCl) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 10% larger than the size of the complex or nanoparticle.
  • concentration of the salt in the composition is no more than about 100 mM (such as no more than about any of 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM, including any ranges between any of these values).
  • the salt is NaCl.
  • the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that does not promote and/or contribute to aggregation of the complex or nanoparticle, or promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 25% (such as no more than about any of 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the complex or nanoparticle.
  • a sugar e.g., sucrose, glucose, or mannitol
  • the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 75% larger than the size of the complex or nanoparticle.
  • the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 50% larger than the size of the complex or nanoparticle.
  • the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 20% larger than the size of the complex or nanoparticle.
  • the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 15% larger than the size of the complex or nanoparticle.
  • the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 10% larger than the size of the complex or nanoparticle.
  • concentration of the sugar in the composition is no more than about 20% (such as no more than about any of 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values).
  • the sugar is sucrose.
  • the sugar is glucose.
  • the sugar is mannitol.
  • the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that does not promote and/or contribute to aggregation of the complex or nanoparticle, or promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 10% (such as no more than about any of 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the complex or nanoparticle.
  • a chemical buffering agent e.g., HEPES or phosphate
  • the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 7.5% larger than the size of the complex or nanoparticle.
  • a chemical buffering agent e.g., HEPES or phosphate
  • the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 5% larger than the size of the complex or nanoparticle.
  • the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 3% larger than the size of the complex or nanoparticle.
  • a chemical buffering agent e.g., HEPES or phosphate
  • the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 1% larger than the size of the complex or nanoparticle.
  • the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that does not promote and/or contribute to the formation of aggregates of the complex or nanoparticles.
  • a chemical buffering agent e.g., HEPES or phosphate
  • the chemical buffering agent is HEPES.
  • the HEPES is added to the composition in the form of a buffer solution comprising HEPES.
  • the solution comprising HEPES has a pH between about 5 and about 9 (such as about any of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9, including any ranges between these values).
  • the composition comprises HEPES at a concentration of no more than about 75 mM (such as no more than about any of 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 mM or less, including any ranges between any of these values).
  • the chemical buffering agent is phosphate.
  • the phosphate is added to the composition in the form of a buffer solution comprising phosphate.
  • the composition does not comprise PBS.
  • the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that does not promote and/or contribute to aggregation of the complex or nanoparticle, or promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the complex or nanoparticle.
  • the composition comprises a cell culture medium (e.g.
  • the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 150% larger than the size of the complex or nanoparticle.
  • the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 100% larger than the size of the complex or nanoparticle.
  • the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 50% larger than the size of the complex or nanoparticle.
  • the composition comprises a cell culture medium (e.g., DMEM or Opti- MEM) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 25% larger than the size of the complex or nanoparticle.
  • the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 10% larger than the size of the complex or nanoparticle.
  • the cell culture medium is DMEM.
  • the composition comprises DMEM at a concentration of no more than about 70% (such as no more than about any of 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10%, or less, including any ranges between any of these values).
  • the composition comprises a carrier protein (e.g., albumin) at a concentration that does not promote and/or contribute to aggregation of the complex or nanoparticle, or promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the complex or nanoparticle.
  • a carrier protein e.g., albumin
  • the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 150% larger than the size of the complex or nanoparticle.
  • the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 100% larger than the size of the complex or nanoparticle.
  • the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 50% larger than the size of the complex or nanoparticle.
  • the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 25% larger than the size of the complex or nanoparticle.
  • the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and/or contributes to the formation of aggregates of the complex or nanoparticles having a size no more than about 10% larger than the size of the complex or nanoparticle.
  • a carrier protein e.g., albumin
  • the carrier protein is albumin.
  • the albumin is human serum albumin.
  • a pharmaceutical composition as described herein is formulated for intravenous, intratumoral, intraarterial, topical, intraocular, ophthalmic, intraportal, intracranial, intracerebral, intracerebroventricular, intrathecal, intravesicular, intradermal, subcutaneous, intramuscular, intranasal, intratracheal, pulmonary, intracavity, or oral administration, or nebulization (NB) or intratracheal instillation.
  • NB nebulization
  • Exemplary dosing frequencies include, but are not limited to, no more than once every three days.
  • the complex or nanoparticle as described herein can be prepared according to the examples or any of the methods described herein.
  • the method involves combining the cell-penetrating peptide with the cargo molecule, thereby forming the genome-editing complex.
  • the method involves preparing the complex comprising a first cellpenetrating peptide and a second cell-penetrating peptide as described above by a) combining the first cell-penetrating peptide and the second cell-penetrating peptide, thereby forming a peptide mixture; b) combining the peptide mixture with the cargo, thereby forming the genome-editing complex.
  • the peptide or the peptide mixture and the cargo molecule can be combined at a molar ratio from about 1:1 to about 100:1 (such as about between about 1:1 and about 50:1, such as about 2:1 to about 50:1), respectively.
  • the method involves mixing a first solution comprising the cargo molecule with a second solution comprising the peptide or peptide mixture to form a third solution, wherein the third solution comprises or is adjusted to comprise i) about 0-5% sucrose, ii) about 0-5% glucose, iii) about 0-50% DMEM, iv) about 0-80 mM NaCl, or v) about 0-20% PBS, and wherein the third solution is incubated to allow formation of the genome-editing complex.
  • the first solution comprises the cargo in sterile water and/or wherein the second solution comprises the peptide or peptide mixture in sterile water.
  • the third solution is adjusted to comprise i) about 0-5% sucrose, ii) about 0-5% glucose, iii) about 0-50% DMEM, iv) about 0-80 mM NaCl, or v) about 0-20% PBS after incubating to form the genome-editing complex.
  • the method further involves a filtration process, wherein the complex is filtered through a pore-sized membrane.
  • the pore has a diameter of at least about 0.1 pm (such as at least about 0.1 pm, 0.15 pm, 0.2 pm, 0.25 pm, 0.3 pm, 0.35 pm, 0.4 pm, 0.45 pm, 0.5pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1.0 pm, 1.1 pm or 1.2 pm).
  • the pore has a diameter of no more about 1.2 pm, 1.0 pm, 0.8 pm, 0.6 pm, 0.5 pm, 0.45 pm, 0.4 pm, 0.35 pm, 0.3 pm, or 0.25 pm.
  • the port has a diameter of about 0.1 m to about 1.2 pm (such as about 0.1 to about 0.8 pm, about 0.2 to about 0.5 pm).
  • the average diameter of the complex or nanoparticle does not change by more than about 10%, and the poly dispersity index does not change by more than about 10%.
  • kit contains vials containing the guide RNA or siRNA, cell-penetrating peptides, other genome-editing molecules and/or other cellpenetrating peptides, combined in one vial or separately in different vials.
  • the kits further comprises materials used for determining the presence of a KRAS mutation (e.g., the various mutations discussed in the application such as G13D, Q61H, A59T, R68S/M, H95D/Q/R, and/or Y96C/D mutation.
  • the cell-penetrating peptides and any molecules are combined accordingly with the appropriate one or more guide RNA to result in complexes or nanoparticles that can be administered to the patient for an effective treatment.
  • kits comprising: 1) a CPP, 2) a guide RNA or a siRNA, and optionally 3) one or more DNA nuclease or polynucleotide encoding the DNA nuclease and 4) a testing kit useful to determine the presence of a KRAS mutation (e.g., a G13D, Q61H, A59T, R68S/M, H95D/Q/R, and/or Y96C/D mutation).
  • the kit further comprises other genome-editing molecules and/or other cell-penetrating peptides.
  • the kit further comprises agents for determining gene expression profiles.
  • the kit further comprises a pharmaceutically acceptable carrier.
  • a kit described herein comprises a) one or more guide RNAs targeting KRAS G12C, G12D and/or G12V as described herein, b) a cell-penetrating peptide, and/or c) an mRNA encoding a CRIS PR-associated endonuclease e.g., unmodified or modified Cas9).
  • a kit described herein comprises a) one or more guide RNAs targeting KRAS G12C, G12D and/or G12V as described herein, b) a cell-penetrating peptide, and/or c) a CRIS PR-associated endonuclease (e.g., unmodified or modified Cas9).
  • a kit described herein comprises a) one or more guide RNAs targeting KRAS G12C, G12D and/or G12V as described herein, b) a cell-penetrating peptide, and/or c) a fusion protein comprising a CRIS PR-associated endonuclease (e.g., unmodified or modified Cas9) and a second enzyme (such as a reversed transcriptase or a nucleobase deaminase enzyme).
  • a CRIS PR-associated endonuclease e.g., unmodified or modified Cas9
  • a second enzyme such as a reversed transcriptase or a nucleobase deaminase enzyme
  • a kit described herein comprises a) one or more guide RNAs targeting KRAS G12C, G12D and/or G12V as described herein, b) a cell-penetrating peptide, and/or c) a polynucleotide encoding a fusion protein comprising a CRIS PR-associated endonuclease (e.g., unmodified or modified Cas9) and a second enzyme (such as a reversed transcriptase or a nucleobase deaminase enzyme).
  • the kit further comprises an agent to assess a mutation of KRAS in an individual.
  • kits described herein may further comprise instructions for using the components of the kit to practice the subject methods (for example instructions for making the pharmaceutical compositions described herein and/or for use of the pharmaceutical compositions).
  • the instructions for practicing the subject methods are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kits or components thereof (i.e., associated with the packaging or sub packaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD- ROM, diskette, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate
  • the various components of the kit may be in separate containers, where the containers may be contained within a single housing, e.g., a box.
  • Embodiment 1 A method of treating a cancer in an individual comprising administering to the individual a complex comprising: a- 1) a cell-penetrating peptide, and a- 2) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, and 273- 341; or b-1) a cell-penetrating peptide, and b-2) a RNAi targeting a mutated KRAS, optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229 and 231-234, further optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual has been subjected to a K
  • Embodiment 2 The method of embodiment 1, wherein the individual comprises a R68, Y96, or A59 mutation, optionally the individual comprises a R68M, Y96D, or A59T mutation on KRAS.
  • Embodiment 3 A method of treating a cancer in an individual comprising administering to the individual a complex comprising: a- 1) a cell-penetrating peptide, and a- 2) a guide RNA targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, and 273- 341; or b-1) a cell-penetrating peptide, and b-2) a RNAi targeting a mutated KRAS, optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229 and 231-234, further optionally wherein the RNAi comprises a siRNA comprising a sequence set forth in any of SEQ ID NOs: 228-229, wherein the individual comprises a R68, Y96
  • Embodiment 4 The method of any one of embodiments 1-3, wherein the cancer is resistant, refractory or recurrent to the KRAS inhibitor, further optionally the individual developed a secondary mutation after the KRAS inhibitor treatment.
  • Embodiment 5 The method of any one of embodiments 2-4, wherein the R68M, Y96D, or A59T mutation is a somatic mutation, and optionally wherein the individual developed the R68M, Y96D, or A59T mutation after the KRAS inhibitor treatment.
  • Embodiment 6 The method of any one of embodiments 1-5, wherein the method further comprises determining whether the individual has the R68M, Y96D, or A59T mutation and/or selecting the individual for treatment due to the presence of the R68M, Y96D, or A59T mutation.
  • Embodiment 7. The method of any one of embodiments 1-6, wherein the individual further comprises a G12C mutation.
  • Embodiment 8 The method of any one of embodiments 1-7, wherein the KRAS inhibitor is a G12C inhibitor.
  • Embodiment 9 The method of embodiment 8, wherein the KRAS inhibitor is sotorasib or adagrasib.
  • Embodiment 10 The method of any one of embodiments 1-7, wherein the KRAS inhibitor is a G12D inhibitor.
  • Embodiment 11 The method of embodiment 10, wherein the G12D inhibitor is MRTX1 133 or RMC-9805.
  • Embodiment 12 The method of any one of embodiments 1-7, wherein the KRAS inhibitor is a G12V inhibitor.
  • Embodiment 13 The method of embodiment 12, wherein the G12V inhibitor is ganetespib.
  • Embodiment 14 The method of any one of embodiments 1-13, wherein the complex is administered at least about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • Embodiment 15 The method of any one of embodiments 1-13, wherein the complex is administered no more about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, or 24 months after the KRAS inhibitor treatment.
  • Embodiment 16 The method of any one of embodiments 1-15, wherein the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).
  • tracrRNA auxiliary trans-activating crRNA
  • Embodiment 17 The method of any one of embodiments 1-16, wherein the nucleotide sequence substantially complementary to a target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34.
  • Embodiment 18 The method of any one of embodiments 1-17, wherein the guide RNA has a length of no more than about 200, 100, 50, 40, 30, 28, or 25 nucleotides.
  • Embodiment 19 The method of any one of embodiments 1-18, wherein the nucleotide sequence is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 3, 19, and 34, optionally wherein nucleotide sequence has the same length as the target sequence.
  • Embodiment 20 The method of any one of embodiments 1-19, wherein the nucleotide is chemically modified.
  • Embodiment 21 The method of any one of embodiments 1-20, wherein the complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease, wherein optionally the DNA nuclease is selected from the group consisting of a CRISPR- associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof.
  • Cas CRISPR- associated protein
  • ZFN zinc finger nuclease
  • TALEN transcription activatorlike effector nuclease
  • meganuclease a variant thereof, a fragment thereof, and a combination thereof.
  • Embodiment 22 The method of embodiment 21, wherein the DNA nuclease comprises a Cas polypeptide, wherein optionally the Cas polypeptide is Cas9.
  • Embodiment 23 The method of any one of embodiments 1-22, wherein the cellpenetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein the cellpenetrating peptide further comprises one or more moieties covalently linked to N-terminus of the cell-penetrating peptide, and wherein the one or more moieties are selected from the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, a linker moiety, and a targeting moiety.
  • the one or more moieties are selected from the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, a linker moiety, and a targeting moiety.
  • Embodiment 25 The method of embodiment 24, wherein the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the cell-penetrating peptide.
  • Embodiment 26 The method of embodiment 24 or embodiment 25, wherein the cellpenetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the cell-penetrating peptide, wherein optionally the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351, 352, 356-366, 378-386, 397-417, and 423-426.
  • Embodiment 27 Embodiment 27.
  • cellpenetrating peptide comprises a linker moiety is selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Ann, Ava, and Ahx.
  • Embodiment 28 The method of any one of embodiments 24-27, wherein the cellpenetrating peptide comprises, from N-terminus, an acetyl group, a targeting moiety and a linker moiety covalently linked to the N-terminus of the cell-penetrating peptide.
  • Embodiment 29 The method of any one of embodiments 1-28, wherein the cellpenetrating peptide further comprises a carbohydrate moiety.
  • Embodiment 30 The method of embodiment 29, wherein the carbohydrate moiety is GalNAc.
  • Embodiment 31 The method of any one of embodiments 1-30, wherein the cellpenetrating peptide is a retro-inverso peptide.
  • Embodiment 32 The method of any one of embodiments 1-31, wherein the cellpenetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-107, 111-117, 153-175 and 272.
  • Embodiment 33 The method of any one of embodiments 1-32, wherein the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1.
  • Embodiment 34 The method of any one of embodiments 21-33, wherein the molar ratio of the cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1.
  • Embodiment 35 The method of any one of embodiments 1-34, further comprising one or more additional guide RNAs comprising different guide sequences.
  • Embodiment 36 The method of embodiment 35, wherein at least two of the two or more guide RNAs target one single KRAS mutation.
  • Embodiment 37 The method of embodiment 36, wherein at least two of the two or more guide RNAs target two or more different KRAS mutations.
  • Embodiment 38 The method of embodiment 36 or 37, wherein at least two of the two or more guide RNAs target G12D, G12V, and/or G12C.
  • Embodiment 39 The method of any one of embodiments 1-38, wherein the average diameter of the complex is between about 10 nm and about 300 nm.
  • Embodiment 40 The method of any one of embodiments 1-39, wherein the complex is in a nanoparticle.
  • Embodiment 41 The method of any one of embodiments 1-40, wherein the method comprises administering two or more complexes, wherein the two or more complexes comprise different guide RNAs that target different KRAS mutations.
  • Embodiment 42 The method of embodiment any one of embodiments 1-41, further comprising administering a second agent to the individual.
  • Embodiment 43 The method of any one of embodiments 1-43, wherein the individual comprises a G12D mutation.
  • Embodiment 44 The method of any one of embodiments 1-44, wherein the individual comprises a G12V mutation.
  • Embodiment 45 The method of any one of embodiments 1, 7-45, wherein the individual does not develop a secondary KRAS mutation in exons 1 and/or 2 after the KRAS treatment.
  • Embodiment 46 The method of any one of embodiments 1-46, wherein the individual is a human.
  • Reagents Lipofectamine 2000, RNAiMAX, TranscriptAid T7 transcription kit, MEGAclear transcription Clean Up kit, GeneArt Genomic Cleavage Detection kit, and Platinum Green Hot Start PCR mix were obtained from Thermo Fisher life Science (France).
  • Antibodies phospho-Akt (Ser 473) (CST, #9271, RRID:AB_329825) and phospho-p44/42 MAPK (Erkl/2) (Thr202/Tyr204) (CST, #4370, RRID:AB_2315112) are from CST.
  • KRASG12C inhibitors Sotorasib (HY-114277) and Adagrasib (HY-130149), were purchased from MedChemExpress [0500] mRNA: CleanCapTM Cas9mRNA (5moU) and CleanCapTM Luc mRNA (5 moU) were obtained for Trilink Biotechnology (USA).
  • sgRNA KRAS sgRNAs targeting KRAS mutation at codon 12 were obtained from Integrated DNA Technologies, Inc. Stock solutions of sgRNAs were solubilized in water, quantified by UV absorbance and stored at -80°C.
  • SiRNA KRAS siRNAs targeting KRAS mutation at codon 12 were obtained from Eurogentec. Stock solutions of siRNAs were solubilized in water, quantified by UV absorbance and stored at -80°C.
  • ADGN Peptides the following ADGN peptides were used
  • Cell lines All cell lines were obtained from the ATCC, MIA-PACA Homozygous for KRAS p.Glyl2Cys (c.34G>T) and H358 Heterozygous for KRAS p.Glyl2Cys (c.34G>T).
  • ADGN/Cas9mRNA/sgRNA complexes were prepared at a 20/1/1 molar ratio with 0.5 pg mRNA: 1.5pg sgRNA and 5% Glucose or DMEM (example for 96 well plates). It is suggested to prepare a minimum volume of complexes for 6 wells of 96 well. Premixed Cas9 mRNA/gRNA were prepared in sterile water at room temperature in a glass vial (1-4 ml). ADGN-peptide solution was added dropwise (1 drop/sec) under magnetic agitation at 400 rpm to obtain a 1:2 ratio, and incubated for 30 min at room temperature or 37°C.
  • ADGN peptide-mRNA:sgRNA complexes were characterized by dynamic light scattering (DLS Malvern Nanosizer) prior transfection. Then the complexes were ready for cell transfection.
  • Transfection protocol The following protocols are reported for 24/48/96 well plate format transfection. Cells should be trypsinized and seeded a day prior transfection Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 2 mM glutamine, 1% antibiotics (streptomycin 10,000 pg/mL, penicillin, 10,000 IU/ mL) and 10% (w/v) fetal calf serum (FCS), at 37°C in a humidified atmosphere containing 5% CO2. 96 well plates seeded with 50,000 cells, the day prior transfection, grown to 60-80% confluence and set up to be at around 70% confluence at the day of transfection.
  • DMEM Dulbecco’s Modified Eagle’s Medium
  • FCS fetal calf serum
  • H358 cells harboring KRAS G12C were exposed to increasing concentrations of sotorasib (up to 10 pM).
  • the minimum concentration of sotorasib (100 nM) was determined as the lowest concentration that suppressed the growth of parental H358 cells.
  • the maximum concentration of sotorasib (2000 nM) corresponds to 100-fold IC50 of the drug.
  • Cells were treated for 2 to 3 weeks, medium and sotorasib were changed every 5 days. Clones resistant to KRAS G12C inhibitors were generated in the course of the treatment.
  • KRAS mutation gene KRAS G12C/Y96D and KRAS G12C/R68M were inserted in pMXs-puro retroviral expression vector (cell biolabs).
  • Retrovirus packing mutated KRAS gene were produced using HEK-293T cells. Viral particles were concentrated using a retrovirus concentration kit (Clontech).
  • H358 and Mia PACA cells (1 x 10 4 ) were infected with the virus packing either KRAS G12C/Y96D or KRAS G12C/R68M g enes an j cultured at 37°C for 72 hrs. The transfected cells were selected with 1.0 pg/mL to 2.0 pg/mL of puromycin for another 72hrs. After puromycin selection, the presence of each KRAS mutation was confirmed by sequencing.
  • H358 and MIA PACA cells were cultured in 96 well plates and treated with reagents (ADGN-122/Sororasib/Adagasib) at 10 different concentrations from 0.1 to 10000 nM, for 72 hours.
  • Cell viability assays were performed using CellTiter Glow kits (Promega) IC50 inhibitory concentration (values were determined by a nonlinear regression curve fit using a variable slope model with normalized response in GraphPad Prism version 8 (GraphPad Software, San Diego, CA).
  • H-358-C1, H-358-C2, H358-C3 KRAS G12C/Y96D
  • H-358-C4 KRAS G12C/R68M
  • H-358-C5 KRAS G12C/A59T
  • Secondary mutations were identified in H358-C3 (KRAS G12C/Y96D), H-358-C4 (KRAS G12C/R68M).
  • H-358-C5 KRAS G12C/A59T.
  • no secondary mutations were identified in the any of the exons in the KRAS gene of H358-C1 and H358-C2.
  • Resistance index was reported in Table 2. Resistance index (RI) was defined as a ratio of the IC50 of each drug for each resistant clone to the respective IC50 of each drug for the parental H-358 cells with KRASG12C.
  • resistant clones showed a Sotorasib resistance index ranging from 128 to 400, with RI >300 for H-358 KRAS G12C/Y96D and H-358 KRAS G12C/R68M clones.
  • H-358 KRAS G12C/R68M, H-358 C2 and H-358 KRAS G12C/Y96D resistant clones showed an Adagrasib resistance index of 21, 58 and 390, respectively.
  • some of the resistant mutations such as A59T and R68M, could be overcome by switching from sotorasib to adagrasib.
  • Y96D resistant mutation is highly resistant to both agents.
  • ADGN-122 nanoparticle corresponds to gRNA34T6 sgRNA/Cas9 mRNA (1/1 molar ratio) associated with ADGN peptide (ADGN-106-Hydro3 peptide) at molar ratio 20/1 (peptide/nucleic acid).
  • ADGN-122 nanoparticles were evaluated on the five stable sotorasib resistant H358 clones. H-358-C1, H-358-C2, H358-C3 (KRAS G12C/Y96D), H-358-C4 (KRAS G12C/R68M). and H-358-C5 (KRAS G12C/A59T).
  • ADGN-122 ADGN/mRNACas9- gRNA complex (from 0.1-10 pM) on day 1.
  • Cell proliferation was analyzed over a period of 5 days using CellTiter Glow kits on GlowMax (Promega) and cytotoxicity was analyzed at 72hr after treatment using CellTiter Glow or MTT assays kits [0519]
  • ADGN-122 using gRNA34T6
  • ADGN-122 inhibits H-358 with KRAS G12C/Y96D which have been identified as major resistance mutation to both Sotorasib and Adagrasib. Accordingly, ADGN-122 overcame resistance associated with secondary mutations.
  • Treatment of H-358 cells harboring KRAS G12C/Y96D with ADGN-122 resulted in IC50 of 11.8 nM, compared with 4484 nM and 7452 nM with sotorasib and adagrasib alone, respectively. See FIG. 4.

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Abstract

La présente demande propose des méthodes de traitement d'un cancer chez des sujets qui ont été soumis à un inhibiteur de KRAS et/ou présentent une mutation secondaire de KRAS par administration de complexes (p. ex. des complexes d'édition de génome ou des complexes à interférence ARN) ou de nanoparticules ciblant spécifiquement un KRAS muté. Des exemples de complexes d'édition de génome ou de nanoparticules comprennent des peptides de pénétration cellulaire et un ARN guide, et éventuellement une nucléase d'ADN (telle que Cas9) ou un polynucléotide codant pour la nucléase d'ADN.
PCT/US2024/019288 2023-03-09 2024-03-09 Compositions de traitement du cancer à mutations de kras et leurs utilisations Pending WO2024187174A2 (fr)

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