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EP-4735598-A2 - NRAS GENE KNOCKOUT FOR TREATMENT OF CANCER

EP4735598A2EP 4735598 A2EP4735598 A2EP 4735598A2EP-4735598-A2

Abstract

The disclosure provides a guide RNA (gRNA) comprising a DNA-binding domain and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease protein-binding domain, wherein the DNA-binding domain is complementary to a target domain from an NRAS gene. The disclosure also provides nucleic acid sequence encoding the gRNA. The disclosure further provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a CRISPR-associated endonuclease and a guide RNA that is complementary to a target domain from an NRAS gene in the subject. Methods of treating cancer comprising administering a pharmaceutical composition comprising: a nucleic acid sequence encoding a guide RNA that is complementary to a target domain from an NRAS gene in the subject; and a nucleic acid sequence encoding a CRISPR-associated endonuclease, are also provided.

Inventors

  • KMIEC, ERIC, B.
  • MASCIARELLI, Sophia
  • SANSBURY, Brett
  • BANAS, Kelly, H.

Assignees

  • Christiana Care Gene Editing Institute, Inc.

Dates

Publication Date
20260506
Application Date
20240701

Claims (20)

  1. 1 . A method of reducing variant NRAS expression or activity in a cancer cell comprising introducing into the cancer cell (a) one or more nucleic acid sequences encoding one or more guide RNAs (gRNAs) that are complementary to one or more target sequences in a variant NRAS gene and (b) a nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the one or more gRNAs hybridize to the variant NRAS gene and the CRISPR-associated endonuclease cleaves the variant NRAS gene, and wherein NRAS expression or activity is reduced in the cancer cell relative to a cancer cell in which the one or more nucleic acid sequences encoding the one or more gRNAs and the nucleic acid sequence encoding the CRISPR-associated endonuclease are not introduced.
  2. 2. The method of claim 1 , wherein the one or more gRNAs comprise a trans-activated small RNA (tracrRNA) and/or a CRISPR RNA (crRNA).
  3. 3. The method of claim 1 or 2, wherein the one or more gRNAs are one or more single guide RNAs.
  4. 4. The method of any one of claims 1 -3, wherein the CRISPR-associated endonuclease is a class 2 CRISPR-associated endonuclease.
  5. 5. The method of claim 4, wherein the class 2 CRISPR-associated endonuclease is Cas12a or Cas9.
  6. 6. The method of any one of claims 1 -5, wherein expression of one or more allele(s) of the variant NRAS gene is reduced in the cancer cell.
  7. 7. The method of any one of claims 1 -6, wherein NRAS activity is reduced in the cancer cell.
  8. 8. The method of any one of claims 1 -7, wherein NRAS expression or activity is not completely eliminated in the cancer cell.
  9. 9. The method of any one of claims 1 -7, wherein NRAS expression or activity is completely eliminated in the cancer cell.
  10. 10. The method of any one of claims 1 -9, wherein expression or activity of wild-type NRAS in a non-cancer cell of a subject that contains the cancer cell is unaffected by the introduction of the one or more nucleic acid sequences of (a) and the nucleic acid sequence of (b).
  11. 1 1 . The method of any one of claims 1 -10, wherein the cancer cell is a lymphoid neoplasm diffuse large B-cell lymphoma, cholangiocarcinoma, uterine carcinosarcoma, kidney chromophobe, uveal melanoma, mesothelioma, adrenocortical carcinoma, thymoma, acute myeloid leukemia, testicular germ cell tumor, rectum adenocarcinoma, pancreatic adenocarcinoma, phenochromocytoma and paraganglioma, esophageal carcinoma, sarcoma, kidney renal papillary cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, glioblastoma multiforme, bladder urothelial carcinoma, colon adenocarcinoma, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, skin cutaneous melanoma, prostate adenocarcinoma, thyroid carcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, brain lower grade glioma, uterine corpus endometrial carcinoma, lung adenocarcinoma, multiple myeloma, breast invasive carcinoma, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, astrocytoma, atypical teratoid/rhabdoid tumor, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, carcinoma of unknown primary, cardiac tumor, medulloblastoma, germ cell tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, CNS germ cell tumor, ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumor, kidney cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, lung cancer (non-small cell, small cell, pleuropulmonary blastoma, tracheobronchial tumor), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, malignant mesothelioma, metastatic cancer, metastatic squamous cell neck cancer with occult primary, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia, plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, or Wilms tumor.
  12. 12. A cancer cell comprising a mutated variant NRAS gene produced by the method of any one of claims 1 -1 1.
  13. 13. A method of reducing variant NRAS expression or activity in a cancer cell comprising introducing into the cancer cell (a) one or more gRNAs that are complementary to one or more target sequences in the variant NRAS gene and (b) a CRISPR-associated endonuclease, whereby the one or more gRNAs hybridize to the variant NRAS gene and the CRISPR-associated endonuclease cleaves the variant NRAS gene, and wherein variant NRAS expression or activity is reduced in the cancer cell relative to a cancer cell in which the one or more gRNAs and the CRISPR- associated endonuclease are not introduced.
  14. 14. The method of claim 13, wherein the one or more gRNAs comprise a tracrRNA and/or a crRNA.
  15. 15. The method of claim 13 or 14, wherein the one or more gRNAs are one or more single guide RNAs.
  16. 16. The method of any one of claims 13-15, wherein the CRISPR-associated endonuclease is a class 2 CRISPR-associated endonuclease.
  17. 17. The method of claim 16, wherein the class 2 CRISPR-associated endonuclease is Cas12a or Cas9.
  18. 18. The method of any one of claims 13-17, wherein expression of one or more allele(s) of the variant NRAS gene is reduced in the cancer cell.
  19. 19. The method of any one of claims 13-18, wherein NRAS activity is reduced in the cancer cell.
  20. 20. The method of any one of claims 13-18, wherein NRAS expression or activity is not completely eliminated in the cancer cell.

Description

TITLE NRAS GENE KNOCKOUT FOR TREATMENT OF CANCER CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application 63/511 ,320, filed June 30, 2023, which is incorporated herein, in its entirety, by reference. SUBMISSION OF SEQUENCE LISTING [0002] The Sequence Listing associated with this application is filed in electronic format via Patent Center and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 13094901701 sequencelisting. xml. The size of the xml file is 11 KB, and the xml file was created on June 27, 2024. FIELD [0003] The present disclosure relates to compositions and methods for knocking out variant NRAS genes found in certain cancers to treat such cancers using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/endonuclease gene editing. BACKGROUND [0004] Cancer is presently one of the leading causes of death in developed nations. A diagnosis of cancer traditionally involves serious health complications. Cancer can cause disfigurement, chronic or acute pain, lesions, organ failure, or even death. Commonly diagnosed cancers include lung cancer, pancreatic cancer, breast cancer, melanoma, lymphoma, carcinoma, sarcoma leukemia, endometrial cancer, colon and rectal cancer, prostate cancer, and bladder cancer. Traditionally, many cancers are treated with surgery, chemotherapy, radiation, or combinations thereof. [0005] Although there have been significant advances in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved only by about 10% in the past 20 years. Cancers, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making treatment extremely difficult. The lack of therapeutic options and increasing resistance to available drugs, creates a huge challenge in cancer therapy. Additionally, the high doses of chemotherapy required at advanced stage causes significant adverse side-effects, deteriorating the quality of life of the patients. [0006] NRAS (also known as NS6, ALPS4, CMNS, and NCMS) encodes a GTPase, which is activated by a guanine nucleotide-exchange factor and inactivated by a GTPase activating protein. Mutations in this gene have been associated with somatic rectal cancer, follicular thyroid cancer, autoimmune lymphoproliferative syndrome, Noonan syndrome, and juvenile myelomonocytic leukemia. [0007] NRAS interacts with the cell membrane and various effector proteins (e.g., Raf and RhoA), which carry out its signaling function through the cytoskeleton and effects on cell adhesion (Fotiadou et al., Mol. Cell. Biol. 27:6742-55 (2007)). Aberrant activation of the RAS pathway is a crucial event in many cancers and is frequently caused by point mutations of hotspot codons located within exon 2 (codons 12 and 13) and exon 3 (codon 61 ). The mutations disrupt intrinsic and RAS-GAP-mediated GTP hydrolysis, leading to constitutive activation and increased affinity of NRAS to the direct effectors, RAFs (ARAF, BRAF, and CRAFJ), RAS-like protein (RAL) guanine nucleotide exchange factors (GEFs), and PI3K (Eisfeld etal., Proc. Natl. Acad. Sci. USA 111 :4179-84 (2014); Vu et al., Pharmacol. Res. 107:111 -16 (2016)). [0008] NRAS and BRAF both play a part in the mitogen-activated protein kinase (MAPK) pathway, which significantly contributes to melanoma development. In physiological conditions, the MAPK pathway is activated by growth factors binding to their surface receptor tyrosine kinase (RTK), and the signal is transmitted through NRAS (Cox et al., Nat. Rev. Drug Discov. 13:828-51 (2014)). However, despite more than three decades of effort by academia and industry, no effective anti-Ras inhibitors have reached the clinic, prompting a widely held perception that Ras oncoproteins are an “undruggable” cancer target. [0009] NRAS mutations are found in melanoma, hematopoietic, and lymphoid tissue malignancies, and sometimes thyroid tumors (Dumaz et al., Cancers (Basel) 11 :1133 (2019)). [0010] Genes encoding members of the two canonical Ras effector families, BRAF and PIK3CA, are also frequently mutationally activated in human cancers (20% and 12%, respectively), supporting the importance of these pathways in driving cancer growth. The serine/threonine kinase B-raf is encoded by the BRAF gene. The most common BRAF alteration observed in human cancers including melanoma is the codon 600 valine to glutamate (V600E) mutation. BRAF V600 is located in the activation segment of the kinase domain close to T599 and S602 which on phosphorylation result in kinase activity. The V600E alteration may mimic the T599/S602 phosphorylation since it was shown that the V600E mutant B-raf has a higher kinase activity than wild type B-raf (Platz et al., Mol. Oncol. 1 :395-405 (2008)). [0011] BRAF mutations are present in approximately 8% of human tumors, but with huge variation in frequency depending on th