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CN-121986166-A - Compositions and methods for genetic modification of CD70

CN121986166ACN 121986166 ACN121986166 ACN 121986166ACN-121986166-A

Abstract

Compositions and methods for editing, e.g., altering, a DNA sequence within a CD70 gene are provided. Compositions and methods for reducing or eliminating CD70 protein expression in a cell are provided. Compositions and methods for immunotherapy are provided.

Inventors

  • M. Ram
  • I. Barwani
  • R. Oliveira
  • LIU BIAO
  • O. Kilic
  • ZHANG BONING

Assignees

  • 因特利亚治疗公司

Dates

Publication Date
20260505
Application Date
20240813
Priority Date
20230814

Claims (20)

  1. 1. An engineered cell comprising a genetic modification within genomic coordinates chr19: 6586002-6591015.
  2. 2. An engineered cell having reduced or eliminated surface expression of CD70 protein relative to an unmodified cell and comprising a genetic modification within genomic coordinates chr19: 6586002-6591015.
  3. 3. The engineered cell of claim 1 or 2, wherein the genetic modification is within genomic coordinates targeted by a CD70 guide RNA comprising a guide sequence of any one of SEQ ID NOs 1-38.
  4. 4. The engineered cell of any one of claims 1-3, having reduced or eliminated CD70 surface expression relative to an unmodified cell, and comprising a genetic modification within any one of the genomic coordinates set forth in table 2A.
  5. 5. The engineered cell of any one of claims 1-4, wherein the genetic modification is within genomic coordinates selected from the group consisting of: chr19:6590121-6590145;chr19:6586002-6586026;chr19:6586003-6586027;chr19:6586013-6586037;chr19:6586357-6586381;chr19:6586365-6586389;chr19:6586376-6586400;chr19:6590988-6591012;chr19:6590991-6591015;chr19:6590862-6590886;chr19:6586396-6586420;chr19:6586372-6586396;chr19:6586371-6586395;chr19:6586360-6586384;chr19:6586355-6586379;chr19:6586268-6586292;chr19:6586259-6586283;chr19:6586256-6586280;chr19:6586142-6586166;chr19:6586141-6586165;chr19:6586135-6586159;chr19:6586128-6586152;chr19:6586127-6586151;chr19:6586126-6586150;chr19:6586121-6586145;chr19:6586120-6586144;chr19:6586096-6586120;chr19:6586055-6586079;chr19:6586029-6586053;chr19:6586023-6586047;chr19:6586312-6586336;chr19:6586151-6586175;chr19:6586145-6586169;chr19:6586100-6586124;chr19:6586030-6586054;chr19:6586028-6586052;chr19:6586395-6586419; And chr19:6586394-6586418.
  6. 6. The engineered cell of any one of claims 1-5, wherein the genetic modification is within genomic coordinates selected from chr19:6590121-6590145 and chr19:6586268-6586292.
  7. 7. The engineered cell of any one of claims 1-6, wherein the genetic modification is within genomic coordinates targeted by a guide RNA comprising the guide sequence of SEQ ID No.1 or 16.
  8. 8. The engineered cell of claim 1 or 2, wherein the genetic modification is within genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs 101-169.
  9. 9. The engineered cell of any one of claims 1, 2, and 8, having reduced or eliminated CD70 surface expression relative to an unmodified cell, and comprising a genetic modification within any one of the genomic coordinates set forth in table 3A.
  10. 10. The engineered cell of any one of claims 1, 2, 8, and 9, wherein the genetic modification is within genomic coordinates selected from the group consisting of: (a) chr19:6590998-6591018;chr19:6590995-6591015;chr19:6590992-6591012;chr19:6590991-6591011;chr19:6590987-6591007;chr19:6590986-6591006;chr19:6590985-6591005;chr19:6590977-6590997;chr19:6590972-6590992;chr19:6590966-6590986;chr19:6590958-6590978;chr19:6590957-6590977;chr19:6590945-6590965;chr19:6590944-6590964;chr19:6590940-6590960;chr19:6590939-6590959;chr19:6590935-6590955;chr19:6590926-6590946;chr19:6590920-6590940;chr19:6590919-6590939;chr19:6590914-6590934;chr19:6590908-6590928;chr19:6590907-6590927;chr19:6590899-6590919;chr19:6590875-6590895;chr19:6590866-6590886;chr19:6590844-6590864;chr19:6590843-6590863;chr19:6586374-6586394;chr19:6586368-6586388;chr19:6586288-6586308;chr19:6586285-6586305;chr19:6586276-6586296;chr19:6586267-6586287;chr19:6586199-6586219;chr19:6586172-6586192;chr19:6586138-6586158;chr19:6586099-6586119; And chr19:6586050-6586070, and (b) chr19:6590875-6590895;chr19:6590844-6590864;chr19:6590843-6590863;chr19:6590835-6590855;chr19:6590104-6590124;chr19:6590096-6590116;chr19:6590095-6590115;chr19:6590094-6590114;chr19:6590093-6590113;chr19:6590087-6590107;chr19:6590084-6590104;chr19:6590083-6590103;chr19:6590078-6590098;chr19:6586368-6586388;chr19:6586299-6586319;chr19:6586267-6586287;chr19:6590842-6590862;chr19:6590139-6590159;chr19:6590138-6590158;chr19:6590135-6590155;chr19:6590079-6590099;chr19:6590077-6590097;chr19:6586412-6586432;chr19:6586404-6586424;chr19:6586403-6586423;chr19:6586396-6586416;chr19:6586396-6586416;chr19:6586395-6586415;chr19:6586388-6586408;chr19:6586380-6586400;chr19:6586379-6586399;chr19:6586375-6586395;chr19:6586369-6586389;chr19:6586367-6586387;chr19:6586360-6586380;chr19:6586359-6586379;chr19:6586120-6586140; and chr19:6586028-6586048.
  11. 11. The engineered cell of any one of claims 1, 2, and 8-10, wherein the genetic modification is within genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs 101, 104, 109, 115, 116, and 123.
  12. 12. The engineered cell of any one of claims 1,2, and 8-11, wherein the genetic modification is within genomic coordinates ：chr19:6590998-6591018;chr19:6590991-6591011;chr19:6590939-6590959;chr19:6590972-6590992;chr19:6590940-6590960; and chr19:6590907-6590927 selected from the group consisting of.
  13. 13. The engineered cell of any one of claims 1, 2, and 8-10, wherein the genetic modification is within genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs 125, 157, 160, 162, 164, and 168.
  14. 14. The engineered cell of any one of claims 1,2, 8-10, and 13, wherein the genetic modification is within genomic coordinates ：chr19:6590875-6590895;chr19:6586396-6586416;chr19:6586388-6586408;chr19:6586379-6586399;chr19:6586369-6586389; and chr19:6586120-6586140 selected from the group consisting of.
  15. 15. A composition comprising a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises: a. A guide sequence selected from SEQ ID NOS.1-38; b. A guide sequence of at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides of a sequence selected from SEQ ID NOS.1-38; c. a guide sequence having at least 85%, 90% or 95% identity to a sequence selected from the group consisting of SEQ ID NOS.1-38; d. a sequence of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in table 2A; e. at least 20, 21, 22, 23 or 24 consecutive nucleotides of the sequence from (d), or F. A guide sequence having at least 85%, 90% or 95% identity to a sequence selected from (d).
  16. 16. A composition comprising a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises: a. A guide sequence selected from SEQ ID NOS.101-169; b. A guide sequence of at least 17, 18, 19 or 20 contiguous nucleotides of a sequence selected from SEQ ID NOS: 101-169; c. A guide sequence having at least 85%, 90% or 95% identity to a sequence selected from the group consisting of SEQ ID NOS: 101-169; d. a sequence of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in table 3A; e. at least 17, 18, 19 or 20 consecutive nucleotides of the sequence from (d), or F. A guide sequence having at least 85%, 90% or 95% identity to a sequence selected from (d).
  17. 17. The composition of claim 15 or 16 for use in altering DNA sequences within a CD70 gene in a cell.
  18. 18. Use of a pharmaceutical composition comprising the composition of claim 15 or 16, or the composition of claim 15 or 16, for inducing a double-strand break or single-strand break in a CD70 gene in a cell, altering a nucleic acid sequence of a CD70 gene in a cell, or reducing expression of a CD70 gene in a cell.
  19. 19. A method of making an engineered human cell having reduced or eliminated surface expression of CD70 protein relative to an unmodified cell, the method comprising contacting the cell with the composition of claim 15 or 16.
  20. 20. A method of reducing surface expression of CD70 protein in a cell relative to an unmodified cell, the method comprising contacting the cell with a composition comprising a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises: a. A guide sequence selected from SEQ ID NOS.1-38; b. A guide sequence of at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides of a sequence selected from SEQ ID NOS.1-38; c. a guide sequence having at least 85%, 90% or 95% identity to a sequence selected from the group consisting of SEQ ID NOS.1-38; d. a sequence of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in table 2A; e. At least 20, 21, 22, 23 or 24 or 25 consecutive nucleotides of the sequence from (d), or F. A guide sequence having at least 85%, 90% or 95% identity to a sequence selected from (d).

Description

Compositions and methods for genetic modification of CD70 I. Cross-reference to related applications The present application is based on the benefit of 35 USC 119 (e) claiming U.S. provisional application Ser. No. 63/519,495 filed on day 2023, 8, 14, and U.S. provisional application Ser. No. 63/610,531 filed on day 2023, 12, 15, each of which is incorporated herein by reference in its entirety. Reference to electronic sequence Listing The present application contains a sequence table submitted electronically in an XML file format and hereby incorporated by reference in its entirety. The XML file was created at month 8 and 12 of 2024, named "01155-0058-00PCT. XML", and was 3,315,072 bytes in size. Disclosure of Invention The present disclosure relates to CRISPR/Cas9 genome editing systems. The present disclosure relates in particular to genetic modification of the CD70 gene. Cluster of differentiation 70 (CD 70) is a cytokine belonging to the Tumor Necrosis Factor (TNF) family (Goodwin et al, 1993). CD70 is a transmembrane protein that is typically transiently expressed on the surface of cd4+ and cd8+ T cells, regulatory T cells (tregs), B cells, antigen presenting cells (such as dendritic cells) and Natural Killer (NK) cells in response to immune activation. CD70 is a known ligand for TNF receptor superfamily protein CD 27. Upon binding to CD27, CD70 triggers intracellular signaling cascades, ultimately leading to a wide variety of outcomes, including T cell expansion and B cell differentiation. While transient CD70 expression plays a key role in promoting normal immune responses, chronic CD70 expression is associated with T cell depletion, a broad term used to describe T cell responses to chronic antigen stimulation (van Gisbergen et al 2009; yang et al 2014). This was first observed in the context of chronic viral infection, but has also been studied in the immune response to tumors. The nature and character of the T cell depletion mechanism may have a crucial impact on checkpoint blockade and the success of adoptive T cell transfer therapies. Thus, there is a need for improved methods and compositions for modifying cells to overcome the problems of chronic CD 70-mediated aberrant immune responses (such as T cell depletion) and to further enhance immune responses. Provided herein are compounds, compositions, systems, and methods for genetically modifying CD 70. For example, compositions and methods for editing a CD70 target sequence (e.g., an insertion, deletion, or substitution of a nucleoside) are provided. Cells having a genetic modification in CD70 are also contemplated. Methods of using the provided compositions to promote immune responses and treat cancer and infectious diseases are also provided. The present disclosure relates to a population of cells comprising cells having a genetic modification in a CD70 sequence as provided herein. The cells may be used in adoptive T cell transfer therapy. The present disclosure relates to compositions and uses of genetically modified cells having a CD70 sequence for use in therapy, such as cancer therapy and immunotherapy. The present disclosure relates to and provides gRNA molecules, CRISPR systems, cells, and methods useful for genome editing of cells. Provided herein is an engineered cell comprising a genetic modification in the CD70 sequence within genomic coordinates chr19: 6586002-6591015. In some embodiments, the present disclosure provides engineered cells that have reduced or eliminated surface expression of CD70 protein due to genetic modification in the CD70 gene. The engineered cell compositions produced by the methods disclosed herein have desirable properties including, for example, reduced or eliminated CD70 protein expression, reduced chronic CD 70-mediated aberrant immune responses (such as T cell depletion), thereby enhancing immune responses. Also disclosed is the use of a cell composition or formulation of any of the preceding embodiments for the manufacture of a medicament for treating a subject. The subject may be a human or animal (e.g., a human or non-human animal, such as a cynomolgus monkey). Preferably, the subject is a human. Also disclosed are any of the foregoing compositions or formulations for generating genetic modifications (e.g., insertions, substitutions, or deletions) of the CD70 gene sequence. In certain embodiments, genetic modifications within the sequence result in changes in the nucleic acid sequence, which prevent translation of the full-length protein prior to genetic modification of the genomic locus, e.g., by making frame shift mutations or nonsense mutations, such that translation terminates prematurely. Genetic modifications may include insertions, substitutions or deletions at splice sites (i.e., splice acceptor sites or splice donor sites) such that aberrant splicing results in frameshift mutations, nonsense mutations or truncated mRNA, thereby prematurely terminating translation. Genetic modifications can also