Compositions and methods for reducing MHC class I within cells

Abstract

Compositions and methods for reducing the expression of MHC class I protein in cells, including genetically modifying MHC class I for use in adoptive cell transfer therapy.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,991, filed on June 16, 2022, and U.S. Provisional Patent Application No. 63 / 494,208, filed on April 4, 2023, each of which is hereby incorporated by reference in its entirety.

[0002] Reference to Electronic Sequence Listing This application includes a sequence listing that was electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML file created on June 13, 2023, is named "01155 - 0054 - 00PCT_SL.xml" and has a size of 14,122,813 bytes.

Summary of the Invention

[0003] The ability to down - regulate MHC class I is essential for many in vivo and ex vivo applications, such as transplantation or the in vitro generation of cell populations that do not activate T cells, when using allogeneic cells (donor - derived cells). In particular, the transfer of allogeneic cells into a subject has attracted great interest in the field of cell therapy. The use of allogeneic cells is limited by the problem of rejection by the recipient's immune cells, which recognize and attack the transplanted cells as foreign substances. To avoid the problem of immune rejection, cell - based therapies have focused on the autologous approach of using the subject's own cells as the cell source for therapy, which is a time - consuming and costly approach.

[0004] Typically, the immune rejection of allogeneic cells is due to the mismatch of major histocompatibility complex (MHC) molecules between the donor and the recipient. In the human population, MHC molecules exist in various forms, including, for example, many genetic variants of any given MHC gene, i.e., alleles encoding different forms of MHC proteins. The main classes of MHC molecules are MHC class I and MHC class II. MHC class I molecules (e.g., HLA-A, HLA-B, and HLA-C in humans) are expressed on all nucleated cells, present antigens, and activate cytotoxic T cells (CD8+ T cells or CTLs). MHC class II molecules (e.g., HLA-DP, HLA-DQ, and HLA-DR in humans) are expressed only on certain types of cells (e.g., B cells, dendritic cells, and macrophages), present antigens, and activate helper T cells (CD4+ T cells or Th cells), and as a result, the helper T cells supply signals to B cells to produce antibodies.

[0005] Minor differences, such as mismatches of MHC alleles between individuals, can activate the recipient's T cells. During T cell development, an individual's T cell repertoire becomes immunotolerant to its own MHC molecules, but T cells that recognize MHC molecules of another individual can persist in the circulating blood, and these T cells are called alloreactive T cells. Alloreactive T cells can be activated, for example, by the presence of cells of another individual expressing MHC molecules in the body, which may cause, for example, graft-versus-host disease and graft rejection.

[0006] Although it is theoretically possible to reduce transplant rejection by completely matching the HLA types between the donor and the recipient, such an approach is logically and realistically difficult considering the diversity of HLA alleles across the entire population. For example, to completely match 10 pairs of alleles (i.e., two pairs of alleles each for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1) is difficult.

[0007] There is interest in methods and compositions for reducing the susceptibility of allogeneic cells to rejection, including, for example, methods and compositions for reducing the expression of MHC proteins in cells to avoid recipient T cell responses. In practice, the ability to genetically modify allogeneic cells for transplantation into a subject has been hampered by the need to edit multiple genes to reduce the expression of all MHC proteins while simultaneously avoiding other detrimental immune responses in the recipient. For example, strategies to deplete MHC class I proteins can reduce CTL activation, while cells lacking MHC class I on their surface are susceptible to lysis by natural killer (NK) cells of the immune system. This is because NK cell activation is regulated by MHC class I-specific inhibitory receptors. Furthermore, several groups have studied the NK protective effects of different MHC class I components, but there are several inconsistencies in this field. See, for example, the Keystone 2022 presentation “Multiplex Base Editing Mitigates CAR-T Cell Allorejection” by Beam Therapeutics-Workshop: Therapeutic Applications session, April 29, 2022 (“Keystone Presentation”). For example, the Keystone Presentation concludes that retention of HLA-A, but not HLA-B or HLA-C, provides protection against NK killing in vitro, while other groups have shown that HLA-C is important for inhibition of NK activity (Xu et al. 2019, Cell Stem Cell 24, 566-578). Furthermore, previous studies have suggested that HLA-E and HLA-G provide some degree of protection, although not complete. Therefore, it has proven difficult to safely reduce or eliminate the expression of MHC class I.

[0008] Gene editing strategies to deplete MHC class II molecules have been found to be particularly difficult, especially in certain types of cells, due to reasons including low editing efficiency and low cell viability, which has hindered their practical use as cell therapies.

[0009] Therefore, there is a need to improve methods and compositions for modifying allogeneic cells in order to address the problem of immune rejection in recipients and the technical problems associated with multiple gene modifications required to generate safer transplantable cells.

[0010] The present disclosure provides engineered human cells with reduced or absent surface expression of HLA-B protein compared to unmodified cells, where the cells are homozygous for HLA-A and HLA-C, or where the cells have reduced or absent surface expression of HLA-A and HLA-B proteins compared to unmodified cells and are homozygous for HLA-C. Thus, the engineered human cells disclosed herein provide a "partial match" approach to allogeneic cell transplantation and MHC class I compatibility issues. The use of cells with reduced or absent expression of HLA-B and homozygous for HLA-A and HLA-C, or cells with reduced or absent expression of HLA-A and HLA-B and homozygous for HLA-C, limits the number of donors required to provide a treatment that covers the majority of the population. This is because in the disclosed partial match approach, only (not two but) one HLA-A allele and (not two but) one HLA-C allele need to match, or only (not two but) one HLA-C allele needs to match. Surprisingly, the engineered human cells disclosed herein with reduced or absent surface expression of only HLA-B protein or both HLA-A and HLA-B proteins compared to unmodified cells show persistence and are protective against NK-mediated rejection, especially when compared to engineered cells with reduced or absent expression of B2M. The present disclosure provides methods and compositions for making such engineered human cells with reduced or eliminated surface expression of only HLA-B protein or both HLA-A and HLA-B proteins compared to unmodified cells, where the cells are homozygous for both HLA-A and HLA-C or homozygous for HLA-C only. In some embodiments, the present disclosure provides engineered human cells, as well as methods and compositions for making the engineered human cells, where the cells further have reduced or eliminated expression of MHC class II proteins at the cell surface, e.g., the cells have a genetic modification in CIITA.In some embodiments, the present disclosure provides for further engineering of cells, including reducing or eliminating the expression of endogenous T cell receptor proteins (e.g., TRAC, TRBC), and introducing exogenous nucleic acids, such as nucleic acids encoding polypeptides that are expressed on the cell surface or polypeptides that are secreted from the cell. Accordingly, the present disclosure provides a flexible platform for genetically engineering human cells for various desired adoptive cell therapy purposes.

[0011] Provided herein are engineered human cells that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the cells are homozygous for HLA-A and homozygous for HLA-C. Also provided are engineered human cells that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from (a) chr6:31354480-31357174 or (b) chr6:31354623-31357108 or 31354497-31357157, and the cells are homozygous for HLA-A and homozygous for HLA-C.

[0012] As used herein, (i) a genetic modification in the HLA-A gene, wherein the genetic modification in the HLA-A gene comprises at least one nucleotide within genomic coordinates selected from chr6:29942854-chr6:29942913 and chr6:29943518-chr6:29943619; and (ii) a genetic modification in the HLA-B gene, wherein the genetic modification in the HLA-B gene comprises at least one nucleotide within genomic coordinates selected from (a) chr6:31354480-31357174 or (b) chr6:31354623-31357108 or 31354497-31357157, and an engineered human cell is provided that has reduced or eliminated surface expression of HLA-A and HLA-B proteins as compared to unmodified cells, and the cell is homozygous for HLA-C.

[0013] As used herein, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from chr6:31355182-31355202; chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355347-31355367; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429.

[0014] As used herein, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from chr6:31355182-31355202; chr6:31355349-31355369; chr6:31355348-31355368; or chr6:31355145-31355165.

[0015] This specification provides engineered human cells that contain gene modifications in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells, where the gene modification comprises at least one nucleotide within genomic coordinates selected from chr6:31355348 - 31355368; chr6:31355349 - 31355369; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355182 - 31355202; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355145 - 31355165; chr6:31355432 - 31355452; chr6:31355340 - 31355360; or chr6:31355414 - 31355434.

[0016] As used herein, there are provided engineered human cells that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31355348-31355368; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355182-31355202; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355145-31355165; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429.

[0017] As used herein, there are provided engineered human cells that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31355348-31355368, chr6:31355349-31355369, chr6:31355192-31355212, chr6:31355347-31355367, chr6:31355340-31355360, chr6:31355409-31355429.

[0018] As used herein, there are provided engineered human cells that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within a genomic locus selected from chr6:31355349-31355369 or chr6:31355348-31355368.

[0019] As used herein, there are provided engineered human cells that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within a genomic locus selected from chr6:31355192-31355212 or chr6:31355347-31355367.

[0020] As used herein, there are provided engineered human cells that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within a genomic locus selected from chr6:31355347-31355367; chr6:31355340-31355360; or chr6:31355409-31355429.

[0021] As used herein, there are provided engineered human cells that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within a genomic locus selected from chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355347-31355367; chr6:31355432-31355452; or chr6:31355340-31355360.

[0022] This specification provides engineered human cells that contain genetic modifications in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells, where the genetic modifications include at least one nucleotide within genomic coordinates selected from chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425; chr6:31355469 - 31355493; chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; chr6:31355441 - 31355465; chr6:31355203 - 31355227; chr6:31356437 - 31356461; chr6:31356426 - 31356450; chr6:31356763 - 31356787; or chr6:31356764 - 31356788.

[0023] This specification provides engineered human cells that contain genetic modifications in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells. The genetic modifications include at least one nucleotide within genomic coordinates selected from chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355361 - 31355385; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355356 - 31355380; chr6:31355366 - 31355390; chr6:31355417 - 31355441; chr6:31357078 - 31357102; chr6:31355460 - 31355484; chr6:31355415 - 31355439; chr6:31355166 - 31355190; chr6:31355378 - 31355402; chr6:31355401 - 31355425; chr6:31356262 - 31356286; chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; chr6:31355441 - 31355465; chr6:31355203 - 31355227; chr6:31356437 - 31356461; chr6:31356426 - 31356450; chr6:31356763 - 31356787; or chr6:31356764 - 31356788.

[0024] As used herein, provided are engineered human cells that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; or chr6:31356426-31356450.

[0025] As used herein, provided are engineered human cells that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; or chr6:31355441-31355465.

[0026] This specification provides engineered human cells that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; or chr6:31355441 - 31355465.

[0027] This specification provides engineered human cells that contain gene modifications in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells. The gene modifications include at least one nucleotide within genomic coordinates selected from chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425; chr6:31355469 - 31355493; chr6:31356262 - 31356286; chr6:31355419 - 31355443; chr6:31355390 - 31355414; chr6:31355369 - 31355393; chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; chr6:31355441 - 31355465; chr6:31355203 - 31355227; chr6:31356437 - 31356461; chr6:31356426 - 31356450; chr6:31356763 - 31356787; chr6:31356764 - 31356788; chr6:31356762 - 31356786; chr6:31355204 - 31355228; chr6:31356436 - 31356460; or chr6:31356767 - 31356791.

[0028] As used herein, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from (a) chr6:31355348-31355368; or (b) chr6:31355390-31355414; chr6:31355417-31355441; or chr6:31356386-31356410.

[0029] As used herein, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31355182-31355202; chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355347-31355367; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429.

[0030] This specification provides engineered human cells that contain gene modifications in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells. The gene modifications include indels, C - to - T substitutions, or A - to - G substitutions within genomic coordinates selected from chr6:31355182 - 31355202; chr6:31355349 - 31355369; chr6:31355348 - 31355368; or chr6:31355145 - 31355165.

[0031] This specification provides engineered human cells that contain gene modifications in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells. The gene modifications include indels, C - to - T substitutions, or A - to - G substitutions within genomic coordinates selected from chr6:31355348 - 31355368; chr6:31355349 - 31355369; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355182 - 31355202; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355145 - 31355165; chr6:31355432 - 31355452; chr6:31355340 - 31355360; or chr6:31355414 - 31355434.

[0032] As used herein, there are provided engineered human cells that contain gene modifications in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the gene modifications include indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr6:31355348-31355368; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355182-31355202; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355145-31355165; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429.

[0033] As used herein, there are provided engineered human cells that contain gene modifications in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the gene modifications include indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr6:31355348-31355368, chr6:31355349-31355369, chr6:31355192-31355212, chr6:31355347-31355367, chr6:31355340-31355360, chr6:31355409-31355429.

[0034] As used herein, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from chr6:31355349-31355369 or chr6:31355348-31355368.

[0035] As used herein, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from chr6:31355192-31355212 or chr6:31355347-31355367.

[0036] As used herein, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from chr6:31355347-31355367; chr6:31355340-31355360; or chr6:31355409-31355429.

[0037] As used herein, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355347-31355367; chr6:31355432-31355452; or chr6:31355340-31355360.

[0038] This specification provides engineered human cells that include gene modifications in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells, where the gene modifications include indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr6:31355182 - 31355202; chr6:31355348 - 31355368; chr6:31355180 - 31355200; chr6:31355145 - 31355165; chr6:31355349 - 31355369; chr6:31355157 - 31355177; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355185 - 31355205; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355190 - 31355210; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355432 - 31355452; chr6:31355340 - 31355360; chr6:31355576 - 31355596; chr6:31355410 - 31355430; chr6:31355419 - 31355439; chr6:31355414 - 31355434; chr6:31355409 - 31355429.

[0039] This specification provides engineered human cells that include gene modification in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells. The gene modification includes indels, C - to - T substitutions, or A - to - G substitutions within genomic coordinates selected from chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425; chr6:31355469 - 31355493; chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; chr6:31355441 - 31355465; chr6:31355203 - 31355227; chr6:31356437 - 31356461; chr6:31356426 - 31356450; chr6:31356763 - 31356787; or chr6:31356764 - 31356788.

[0040] This specification provides engineered human cells that contain genetic modifications in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, where the genetic modifications include indels, C-to-T substitutions, or A-to-G substitutions within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355361-31355385; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355356-31355380; chr6:31355366-31355390; chr6:31355417-31355441; chr6:31357078-31357102; chr6:31355460-31355484; chr6:31355415-31355439; chr6:31355166-31355190; chr6:31355378-31355402; chr6:31355401-31355425; chr6:31356262-31356286; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; or chr6:31356764-31356788.

[0041] As used herein, there are provided engineered human cells that contain gene modifications in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the gene modifications include indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; or chr6:31356426-31356450.

[0042] As used herein, there are provided engineered human cells that contain gene modifications in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the gene modifications include indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385.

[0043] This specification provides engineered human cells that contain gene modifications in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, where the gene modifications include indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425; chr6:31355469 - 31355493; chr6:31356262 - 31356286; chr6:31355419 - 31355443; chr6:31355390 - 31355414; chr6:31355369 - 31355393; chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; chr6:31355441 - 31355465; chr6:31355203 - 31355227; chr6:31356437 - 31356461; chr6:31356426 - 31356450; chr6:31356763 - 31356787; chr6:31356764 - 31356788; chr6:31356762 - 31356786; chr6:31355204 - 31355228; chr6:31356436 - 31356460; or chr6:31356767 - 31356791.

[0044] As used herein, there are provided engineered human cells that include gene modification in the HLA-B gene and have reduced or eliminated surface expression of HLA-B protein as compared to unmodified cells, wherein the gene modification includes an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from (a) chr6:31355348-31355368; or (b) chr6:31355390-31355414; chr6:31355417-31355441; or chr6:31356386-31356410.

[0045] As used herein, there is provided a method of making an engineered human cell that has reduced or eliminated surface expression of HLA-B protein as compared to unmodified cells, wherein the cell is homozygous for HLA-A and homozygous for HLA-C, and the cell is contacted with a composition comprising (a) an HLA-B guide RNA comprising (i) a guide sequence selected from SEQ ID NOs: 1-91 and 101-185; or (ii) at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1-91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 101-185; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or (v) a guide sequence that is complementary to at least 17, 18, 19, or 20 consecutive nucleotides of a genomic region listed in Table 2, or a guide sequence that is complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a genomic region listed in Table 3; or (vi) a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from (v); and optionally (b) an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0046] This specification provides a method for producing engineered human cells having reduced or eliminated surface expression of HLA-A and HLA-B proteins as compared to unmodified cells, where the cells are homozygous for HLA-C, and the cells are contacted with a first composition comprising: (a) an HLA-A guide RNA comprising a guide sequence selected from (i) SEQ ID NOs: 301-590; or (ii) at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301-428 and 463-511; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 429-462 and 512-590; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301-428 and 463-511; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 429-462 and 512-590; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Tables 4-7; or (v) a guide sequence complementary to at least 17, 18, 19, or 20 consecutive nucleotides of a genomic region listed in Table 4, Table 5B, or Table 6; or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a genomic region listed in Table 5A or Table 7; or (vi) a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from (v); and optionally (b) a first RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent; and the cells are contacted with a first composition comprising: (a) a guide sequence selected from (i) SEQ ID NOs: 1-91 and 101-185; or (ii) at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1-91;a guide sequence that is at least 95%, 90%, 85%, 80%, 75% or 70% identical to a sequence selected from SEQ ID NOs: 101 to 185; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or (v) a guide sequence complementary to at least 17, 18, 19 or 20 consecutive nucleotides of the genomic region listed in Table 2, or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of the genomic region listed in Table 3; or (vi) a guide sequence that is at least 95%, 90%, 85%, 80%, 75% or 70% identical to a sequence selected from (v); and optionally (b) contacting the cell with a second composition comprising an HLA-B guide RNA and, optionally, an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent;.

[0047] The present specification provides a method for reducing the surface expression of HLA-B protein in human cells as compared to non-modified cells, the method comprising contacting the cells with a composition comprising (a) an HLA-B guide RNA comprising (i) a guide sequence selected from SEQ ID NOs: 1 to 91 and 101 to 185; or (ii) at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1 to 91; or at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101 to 185; or (iii) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from SEQ ID NOs: 1 to 91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75% or 70% identical to a sequence selected from SEQ ID NOs: 101 to 185; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or (v) a guide sequence complementary to at least 17, 18, 19 or 20 consecutive nucleotides of a genomic region listed in Table 2, or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a genomic region listed in Table 3; or (vi) a guide sequence that is at least 95%, 90% or 85%, 80%, 75% or 70% identical to a sequence selected from (v); and optionally (b) an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0048] This specification provides a method for reducing the surface expression of HLA-A and HLA-B proteins in human cells as compared to unmodified cells, the method comprising contacting the cells with a first composition comprising: (a) an HLA-A guide RNA comprising a guide sequence selected from (i) SEQ ID NOs: 301 to 590; or (ii) at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301 to 428 and 463 to 511; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 429 to 462 and 512 to 590; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301 to 428 and 463 to 511; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 429 to 462 and 512 to 590; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Tables 4 to 7; or (v) a guide sequence complementary to at least 17, 18, 19, or 20 consecutive nucleotides of a genomic region listed in Table 4, Table 5B, or Table 6; or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a genomic region listed in Table 5A or Table 7; or (vi) a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from (v); and optionally (b) a first RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent; and contacting the cells with a second composition comprising: (a) a guide sequence selected from (i) SEQ ID NOs: 1 to 91 and 101 to 185; or (ii) at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1 to 91; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101 to 185; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1 to 91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 101 to 185;or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or (v) a guide sequence complementary to at least 17, 18, 19, or 20 consecutive nucleotides of a genomic region listed in Table 2, or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a genomic region listed in Table 3; or (vi) a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from (v); and contacting a cell with a second composition comprising the HLA-B guide RNA and optionally (b) an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent; and contacting a cell with a composition comprising;

[0049] Provided herein is a method of administering an engineered cell to a recipient subject in need thereof, the method comprising: (a) determining the HLA-A and HLA-C alleles of the recipient subject; (b) selecting an engineered cell or cell population of any one of the preceding embodiments, or an engineered cell or cell population generated by the method of any one of the preceding embodiments, wherein the engineered cell comprises at least one of the same HLA-A or HLA-C alleles as the recipient subject; and (c) administering the selected engineered cell to the recipient subject.

[0050] Provided herein is a method of administering an engineered cell to a recipient subject in need thereof, the method comprising: (a) determining the HLA-C allele of the recipient subject; (b) selecting an engineered cell or cell population of any one of the preceding embodiments, or an engineered cell or cell population generated by the method of any one of the preceding embodiments, wherein the engineered cell comprises at least one of the same HLA-C alleles as the recipient subject; and (c) administering the selected engineered cell to the recipient subject.

[0051] Further embodiments are provided and described throughout the claims and the drawings.

Brief Description of the Drawings

[0052]

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[0053] The present disclosure provides engineered human cells, as well as methods and compositions for genetically modifying human cells to produce engineered human cells useful, for example, in adoptive cell transfer (ACT) therapy. The present disclosure provides engineered human cells having reduced or eliminated surface expression of HLA-B protein compared to unmodified cells, wherein the cells are homozygous for HLA-A and homozygous for HLA-C. Further, the present disclosure provides engineered human cells having reduced or eliminated surface expression of HLA-A and HLA-B proteins compared to unmodified cells, wherein the cells are homozygous for HLA-C. Thus, the engineered human cells disclosed herein provide a "partial match" solution to the problems associated with allogeneic cell transplantation.

[0054] In some embodiments, the present disclosure provides engineered cells having reduced or eliminated surface expression of HLA-B protein as a result of genetic modification in the HLA-B gene, wherein the cells are homozygous for HLA-A and HLA-C. In some embodiments, the present disclosure provides compositions and methods for reducing or eliminating the expression of HLA-B protein and compositions and methods for reducing the susceptibility of cells to immune rejection reactions, compared to non-engineered cells. In some embodiments, engineered human cells having reduced or eliminated surface expression of HLA-B protein, compared to non-engineered cells, are less susceptible to lysis by NK cells, which is a problem observed with other approaches for reducing or eliminating the expression of MHC class I proteins. In some embodiments, the methods and compositions comprise reducing or eliminating the surface expression of HLA-B protein by genetically modifying HLA-B with a gene editing system and inserting an exogenous nucleic acid encoding a targeting receptor or other polypeptide (expressed on the cell surface or secreted) into the cell by genetic modification. Engineered cell compositions produced by the methods disclosed herein have desirable properties including, for example, reduced or eliminated expression of HLA-B, reduced immunogenicity in vitro and in vivo, increased survival rates, and increased genetic compatibility with a greater number of transplant recipients.

[0055] In some embodiments, the present disclosure provides engineered cells having reduced or eliminated surface expression of HLA-A and HLA-B proteins as a result of genetic modification in the HLA-A and HLA-B genes, wherein the cells are homozygous for HLA-C. In some embodiments, the present disclosure provides compositions and methods for reducing or eliminating the expression of HLA-A and HLA-B proteins, and compositions and methods for reducing the susceptibility of cells to immune rejection reactions, compared to non-engineered cells. In some embodiments, engineered human cells having reduced or eliminated surface expression of HLA-A and HLA-B proteins, compared to non-engineered cells, are less susceptible to lysis by NK cells, which is a problem observed with other approaches for reducing or eliminating the expression of MHC class I proteins. In some embodiments, the methods and compositions reduce or eliminate the surface expression of HLA-A and HLA-B proteins by genetically modifying HLA-A and HLA-B with a gene editing system and inserting into the cells, by genetic modification, an exogenous nucleic acid encoding a targeting receptor or other polypeptide (expressed on the cell surface or secreted). Engineered cell compositions produced by the methods disclosed herein have desirable properties including, for example, reduced or eliminated surface expression of HLA-A and HLA-B proteins, reduced immunogenicity in vitro and in vivo, increased survival rates, and increased genetic compatibility with a greater number of transplantation recipients.

[0056] The terms "about" or "approximately" mean an acceptable error for a particular value sought by a person skilled in the art, which is in part determined by the method of measuring or determining that value, or by the degree of variation that does not substantially affect the characteristics of the matter described, or is within the tolerances in the art, for example, within 10%, 5%, 2% or 1%. Accordingly, unless otherwise specified, the numerical parameters set forth in the following specification and the appended claims are approximate values that may vary depending on the desired characteristics to be obtained. Each numerical parameter should be construed at least in light of the number of significant digits reported and by applying ordinary rounding techniques, not as attempts to limit the application of the doctrine of equivalents to the claims.

[0057] In this specification, the following numbered embodiments are provided.

[0058] Embodiment 1 is an engineered human cell that includes gene modification in the HLA-A gene and gene modification in the HLA-B gene and has reduced or eliminated surface expression of HLA-A and HLA-B proteins compared to unmodified cells, wherein the cell is homozygous for HLA-C.

[0059] Embodiment 2 involves gene modification in the HLA-A gene and gene modification in the HLA-B gene, and is an engineered human cell having reduced or eliminated surface expression of HLA-A and HLA-B proteins as compared to unmodified cells. (i) The gene modification in the HLA-A gene includes at least one nucleotide within genomic coordinates selected from (a) chr6:29942854-chr6:29942913 and chr6:29943518-chr6:29943619; and (b) chr6:29942540-29945459. (ii) The gene modification in the HLA-B gene includes at least one nucleotide within genomic coordinates selected from (a) chr6:31354480-31357174 or (b) chr6:31354497-31357157. The cell is homozygous for HLA-C.

[0060] Embodiment 3 involves gene modification in the HLA-B gene and is an engineered human cell having reduced or eliminated surface expression of HLA-B protein as compared to unmodified cells. The cell is homozygous for HLA-A and homozygous for HLA-C.

[0061] Embodiment 4 involves gene modification in the HLA-B gene and is an engineered human cell having reduced or eliminated surface expression of HLA-B protein as compared to unmodified cells. The gene modification includes at least one nucleotide within genomic coordinates selected from (a) chr6:31354480-31357174 or (b) chr6:31354497-31357157. The cell is homozygous for HLA-A and homozygous for HLA-C.

[0062] Embodiment 5 is the engineered human cell according to any one of Embodiments 1 to 4, wherein the cell has reduced or eliminated expression of at least one HLA-B allele selected from HLA-B7, HLA-B8, HLA-B35, HLA-B40, HLA-B44, HLA-B15, HLA-B14, HLA-B18, and HLA-B51.

[0063] Embodiment 6 is the engineered human cell according to any one of Embodiments 1, 2, or 5, wherein the cell has reduced or eliminated expression of at least one HLA-A allele selected from HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A29, HLA-A26, HLA-A33, and HLA-A24.

[0064] Embodiment 7 is the engineered cell according to any one of Embodiments 1 to 6, wherein the genetic modification in the HLA-B gene comprises at least one nucleotide within the genomic coordinates selected from (a) chr6:31355182-31355596 or (b) chr6:31355203-31356461.

[0065] Embodiment 8 is the engineered cell according to any one of Embodiments 1 to 7, wherein the gene modification in the HLA-B is (a) chr6:31355182-31355202; chr6:31355348-31355368; chr6:31355180-31355200; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31355157-31355177; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355185-31355205; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355190-31355210; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355576-31355596; chr6:31355410-31355430; chr6:31355419-31355439; chr6:31355414-31355434; or chr6:31355409-31355429; or (b) chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355415-31355439; chr6:31355378-31355402; chr6:31355166-31355190;Comprising at least one nucleotide within genomic coordinates selected from chr6:31355401-31355425; ch6:31355469-31355493; chr6:31356262-31356286; chr6:31355419-31355443; chr6:31355390-31355414; chr6:31355369-31355393; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; chr6:31356764-31356788; chr6:31356762-31356786; chr6:31355204-31355228; chr6:31356436-31356460; or chr6:31356767-31356791.;

[0066] Embodiment 9 is the engineered cell according to any one of Embodiments 1 to 8, wherein the gene modification in HLA-B comprises at least one nucleotide within a genomic locus selected from chr6:31355182-31355202; chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355347-31355367; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429.

[0067] Embodiment 10 is the engineered cell according to any one of Embodiments 1 to 9, wherein the gene modification in HLA-B comprises at least one nucleotide within a genomic locus selected from chr6:31355182-31355202; chr6:31355349-31355369; chr6:31355348-31355368; or chr6:31355145-31355165.

[0068] Embodiment 11 is the engineered cell according to any one of Embodiments 1 to 10, and the gene modification in the HLA-B includes at least one nucleotide within the genomic coordinates selected from chr6:31355348-31355368; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355182-31355202; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355145-31355165; chr6:31355432-31355452; chr6:31355340-31355360; or chr6:31355414-31355434.

[0069] Embodiment 12 is the engineered cell according to any one of Embodiments 1 to 11, and the gene modification in the HLA-B includes at least one nucleotide within the genomic coordinates selected from chr6:31355348-31355368; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355182-31355202; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355145-31355165; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429.

[0070] Embodiment 13 is the engineered cell according to any one of Embodiments 1 to 12, and the gene modification in the HLA-B includes at least one nucleotide within the genomic coordinates selected from chr6:31355348-31355368, chr6:31355347-31355367, chr6:31355349-31355369, chr6:31355192-31355212, chr6:31355340-31355360, chr6:31355409-31355429.

[0071] Embodiment 14 is the engineered cell according to any one of Embodiments 1 to 13, and the gene modification in the HLA-B includes at least one nucleotide within a genomic locus selected from (i) chr6:31355349-31355369 or chr6:31355348-31355368; (ii) chr6:31355192-31355212 or chr6:31355347-31355367; (iii) chr6:31355347-31355367; chr6:31355340-31355360; or chr6:31355409-31355429; or (iv) chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355347-31355367; chr6:31355432-31355452; or chr6:31355340-31355360.

[0072] Embodiment 15 is an engineered cell according to any one of Embodiments 1 to 14, wherein the genetic modification in HLA-B comprises at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355415-31355439; chr6:31355378-31355402; chr6:31355166-31355190; chr6:31355401-31355425; ch6:31355469-31355493; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; or chr6:31356764-31356788.

[0073] Embodiment 16 is the engineered cell according to any one of Embodiments 1 to 15, wherein the genetic modification in HLA-B comprises at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355361-31355385; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355356-31355380; chr6:31355366-31355390; chr6:31355417-31355441; chr6:31357078-31357102; chr6:31355460-31355484; chr6:31355415-31355439; chr6:31355166-31355190; chr6:31355378-31355402; chr6:31355401-31355425; chr6:31356262-31356286; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; or chr6:31356764-31356788.

[0074] Embodiment 17 is the engineered cell according to any one of Embodiments 1 to 16, wherein the gene modification in HLA-B comprises at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; ch6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; or chr6:31356426-31356450.

[0075] Embodiment 18 is the engineered cell according to any one of Embodiments 1 to 17, wherein the gene modification in HLA-B comprises at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; ch6:31355491-31355515; chr6:31355361-31355385; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; or chr6:31355441-31355465.

[0076] Embodiment 19 is the engineered cell according to any one of Embodiments 1 to 18, and the gene modification in the HLA-B includes at least one nucleotide within the genomic coordinates selected from chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; or chr6:31355441-31355465.

[0077] Embodiment 20 is the engineered cell according to any one of Embodiments 1 to 19, and the gene modification in the HLA-B includes at least one nucleotide within the genomic coordinates selected from (a) chr6:31355348-31355368; or (b) chr6:31355390-31355414; chr6:31355417-31355441; or chr6:31356386-31356410.

[0078] Embodiment 21 is the engineered cell according to any one of Embodiments 1 to 2 and 5 to 20, and the gene modification in the HLA-A includes at least one nucleotide within the genomic coordinates selected from chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; and chr6:29942883-29942903.

[0079] Embodiment 22 is an engineered cell according to any one of Embodiments 1 to 2 and 5 to 21, wherein the gene modification in the HLA-A comprises at least one nucleotide within genomic coordinates selected from chr6:29942891-29942915; chr6:29942609-29942633; chr6:29942864-29942884; chr6:29944266-29944290; chr6:29942889-29942913; chr6:29942891-29942915chr6:29944471-29944495; chr6:29944470-29944494.

[0080] Embodiment 23 is an engineered cell according to any one of Embodiments 1 to 2 and 5 to 22, wherein the gene modification in the HLA-A comprises at least one nucleotide within genomic coordinates selected from chr6:29942891-29942915; chr6:29942609-29942633.

[0081] Embodiment 24 is an engineered human cell that includes gene modification in the HLA - B gene and has reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, and the gene modification is (a) chr6:31355348 - 31355368; or chr6:31355347 - 31355367; chr6:31355182 - 31355202; chr6:31355180 - 31355200; chr6:31355145 - 31355165; chr6:31355349 - 31355369; chr6:31355157 - 31355177; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355185 - 31355205; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355190 - 31355210; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355178 - 31355198; chr6:31355432 - 31355452; chr6:31355340 - 31355360; chr6:31355576 - 31355596; chr6:31355410 - 31355430; chr6:31355419 - 31355439; chr6:31355414 - 31355434; or chr6:31355409 - 31355429; or (b) chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; chr6:31355441 - 31355465; chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385;Genomic coordinates selected from chr6:31355356-31355380;chr6:31355460-31355484;chr6:31357078-31357102;chr6:31355417-31355441;chr6:31355366-31355390;chr6:31355415-31355439;chr6:31355378-31355402;chr6:31355166-31355190;chr6:31355401-31355425;ch6:31355469-31355493;chr6:31356262-31356286;chr6:31355419-31355443;chr6:31355390-31355414;chr6:31355369-31355393;chr6:31355203-31355227;chr6:31356437-31356461;chr6:31356426-31356450;chr6:31356763-31356787;chr6:31356764-31356788;chr6:31356762-31356786;chr6:31355204-31355228;chr6:31356436-31356460; or chr6:31356767-31356791, including indels, C to T substitutions, or A to G substitutions within the genomic coordinates.;

[0082] Embodiment 25 is an engineered human cell having reduced or eliminated surface expression of HLA-A and HLA-B proteins, wherein (i) (a) chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; chr6:29944026-29944046; chr6:29934330-29934350, chr6:29943115-29943135, chr6:29943135-29943155, chr6:29943140-29943160, chr6:29943590-29943610, chr6:29943824-29943844, chr6:29943858-29943878, chr6:29944478-29944498 and chr6:29944850-29944870; or (b) an indel, C to T substitution or A to G substitution within genomic coordinates selected from chr6:29942891-29942915; chr6:29942609-29942633; chr6:29944266-29944290; chr6:29942889-29942913; chr6:29944471-29944495; and chr6:29944470-29944494 in the HLA-A gene, and (ii) (a) chr6:31355348-31355368; or chr6:31355347-31355367; chr6:31355182-31355202; chr6:31355180-31355200; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31355157-31355177; chr6:31356381-31356401;chr6: 31356380 - 31356400; chr6: 31355204 - 31355224; chr6: 31355205 - 31355225; chr6: 31355185 - 31355205; chr6: 31355191 - 31355211; chr6: 31355192 - 31355212; chr6: 31355190 - 31355210; chr6: 31355193 - 31355213; chr6: 31355198 - 31355218; chr6: 31355320 - 31355340; chr6: 31355319 - 31355339; chr6: 31355178 - 31355198; chr6: 31355432 - 31355452; chr6: 31355340 - 31355360; chr6: 31355576 - 31355596; chr6: 31355410 - 31355430; chr6: 31355419 - 31355439; chr6: 31355414 - 31355434; and chr6: 31355409 - 31355429; or (b) chr6: 31355221 - 31355245; chr6: 31355222 - 31355246; chr6: 31355205 - 31355229; chr6: 31355446 - 31355470; chr6: 31356425 - 31356449; chr6: 31355441 - 31355465; chr6: 31356777 - 31356801; chr6: 31355492 - 31355516; chr6: 31355379 - 31355403; chr6: 31355491 - 31355515; chr6: 31355361 - 31355385; chr6: 31355356 - 31355380; chr6: 31355460 - 31355484; chr6: 31357078 - 31357102; chr6: 31355417 - 31355441; chr6: 31355366 - 31355390; chr6: 31355415 - 31355439; chr6: 31355378 - 31355402; chr6: 31355166 - 31355190; chr6: 31355401 - 31355425; ch6: 31355469 - 31355493; chr6: 31356262 - 31356286; chr6: 31355419 - 31355443; chr6: 31355390 - 31355414;A gene modification in the HLA - B gene, including an indel, a C - to - T substitution, or an A - to - G substitution within genomic coordinates selected from chr6:31355369 - 31355393; chr6:31355203 - 31355227; chr6:31356437 - 31356461; chr6:31356426 - 31356450; chr6:31356763 - 31356787; chr6:31356764 - 31356788; chr6:31356762 - 31356786; chr6:31355204 - 31355228; chr6:31356436 - 31356460; or chr6:31356767 - 31356791.

[0083] Embodiment 26 is the engineered cell according to any one of Embodiments 24 or 25, wherein the gene modification in the HLA - B is (a) chr6:31355348 - 31355368; or (b) an indel, a C - to - T substitution, or an A - to - G substitution within genomic coordinates selected from chr6:31355390 - 31355414; chr6:31355417 - 31355441; or chr6:31355390 - 31355414.

[0084] Embodiment 27 is the engineered cell according to any one of Embodiments 24 - 26, wherein the gene modification in the HLA - A or the gene modification in the HLA - B comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides within the genomic coordinates, or the gene modification comprises at least 5 consecutive nucleotides within the genomic coordinates.

[0085] Embodiment 28 is the engineered cell according to any one of Embodiments 24 - 27, wherein the gene modification in the HLA - A or the gene modification in the HLA - B comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates.

[0086] Embodiment 29 is the engineered cell according to any one of Embodiments 24 to 28, wherein the gene modification in HLA-A or the gene modification in HLA-B includes at least one C-to-T substitution or at least one A-to-G substitution within the genomic coordinates.

[0087] Embodiment 30 is the engineered cell according to any one of Embodiments 1 to 29, wherein the HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within the genomic coordinates selected from chr6:31355348-31355368; or chr6:31355347-31355367; chr6:31355182-31355202; chr6:31355348-31355368; chr6:31355180-31355200; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31355157-31355177; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355185-31355205; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355190-31355210; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355576-31355596; chr6:31355410-31355430; chr6:31355419-31355439; chr6:31355414-31355434; chr6:31355409-31355429.

[0088] Embodiment 31 is an engineered cell according to any one of Embodiments 1 to 30, and the HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within the genomic coordinates selected from chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355415-31355439; chr6:31355378-31355402; chr6:31355166-31355190; chr6:31355401-31355425; ch6:31355469-31355493; chr6:31356262-31356286; chr6:31355419-31355443; chr6:31355390-31355414; chr6:31355369-31355393; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; chr6:31356764-31356788; chr6:31356762-31356786; chr6:31355204-31355228; chr6:31356436-31356460 or chr6:31356767-31356791.

[0089] Embodiment 32 is the engineered cell according to any one of Embodiments 1 to 2, 5 to 23, and 25 to 31, and HLA-A expression is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 consecutive nucleotides within the genomic coordinates selected from chr6:29942891-29942915; chr6:29942609-29942633; chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; chr6:29944026-29944046; chr6:29934330-29934350, chr6:29943115-29943135, chr6:29943135-29943155, chr6:29943140-29943160, chr6:29943590-29943610, chr6:29943824-29943844, chr6:29943858-29943878, and chr6:29944478-29944498, chr6:29944850-29944870.

[0090] Embodiment 33 is the engineered cell according to any one of Embodiments 1, 2, 5 to 23, and 25 to 32, wherein HLA-A expression is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 consecutive nucleotides within the genomic coordinates selected from chr6:29942891-29942915; chr6:29942609-29942633; chr6:29944266-29944290; chr6:29942889-29942913; chr6:29944471-29944495 and chr6:29944470-29944494.

[0091] Embodiment 34 is the engineered cell according to any one of Embodiments 1, 2, 5 to 23, and 25 to 33, wherein HLA-A expression is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 consecutive nucleotides within the genomic coordinates selected from chr6:29942891-29942915 or chr6:29942609-29942633.

[0092] Embodiment 35 is the engineered cell according to any one of Embodiments 30 to 34, wherein the HLA-A genomic target sequence or the HLA-B genomic target sequence comprises at least 10 consecutive nucleotides within the genomic coordinates.

[0093] Embodiment 36 is the engineered cell according to any one of Embodiments 30 to 35, wherein the HLA-A genomic target sequence or the HLA-B genomic target sequence comprises at least 15 consecutive nucleotides within the genomic coordinates.

[0094] Embodiment 37 is the engineered cell according to any one of Embodiments 30 to 36, wherein the HLA-A genomic target sequence or the HLA-B genomic target sequence comprises at least 17, 18, 19, or 20 consecutive nucleotides within the genomic coordinates.

[0095] Embodiment 38 is an engineered cell according to any one of Embodiments 30 to 36, wherein the HLA-A genomic target sequence or the HLA-B genomic target sequence comprises at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides within the genomic coordinates.

[0096] Embodiment 39 is an engineered cell according to any one of Embodiments 1 to 38, wherein the cell is homozygous for HLA-C.

[0097] Embodiment 40 is an engineered cell according to any one of Embodiments 1 to 39, wherein the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C*07:02; HLA-C*07:01; HLA-C*05:01; HLA-C*04:01; HLA-C*03:04; HLA-C*06:02; HLA-C*08:02; HLA-C*08:01; HLA-C*03:02; HLA-C*06:02; HLA-C*16:01; HLA-C*12:03; HLA-C*04:01; HLA-C*15:02; HLA-C*07:01; HLA-C*03:04; HLA-C*12:03; HLA-C*02:10; HLA-C*05:01; HLA-C*12:02; HLA-C*14:02; HLA-C*06:02; HLA-C*04:01; HLA-C*03:03; HLA-C*07:04; HLA-C*07:04; HLA-C*04:01; HLA-C*17:01; HLA-C*01:02 and HLA-C*02:02.

[0098] Embodiment 41 is an engineered cell according to any one of Embodiments 1 to 40, wherein the HLA-C allele is HLA-C*07:02.

[0099] Embodiment 42 is an engineered cell according to any one of Embodiments 1 to 40, wherein the HLA-C allele is HLA-C*07:01.

[0100] Embodiment 43 is the engineered cell according to any one of Embodiments 1 to 40, and the HLA-C allele is HLA-C*05:01.

[0101] Embodiment 44 is the engineered cell according to any one of Embodiments 1 to 40, and the HLA-C allele is HLA-C*04:01.

[0102] Embodiment 45 is the engineered cell according to any one of Embodiments 1 to 40, and the HLA-C allele is HLA-C*06:02.

[0103] Embodiment 46 is the engineered cell according to any one of Embodiments 3 to 24 and 26 to 45, the engineered cell is homozygous for HLA-A, and the HLA-A allele is any one of the following HLA-A alleles: HLA-A*02:01; HLA-A*01:01; HLA-A*03:01; HLA-A*11:01; HLA-A*26:01; HLA-A*68:01; HLA-A*29:02; HLA-A*31:01; HLA-A*32:01; HLA-A*30:02; HLA-A*25:01; HLA-A*33:01; HLA-A*02:02; HLA-A*74:01; HLA-A*02:02; HLA-A*29:01; HLA-A*02:03; HLA-A*02:05; HLA-A*24:07; HLA-A*11:02; HLA-A*36:01; HLA-A*02:22; HLA-A*34:02; HLA-A*01:03; HLA-A*24:02; HLA-A*02:07; HLA-A*23:01; HLA-A*30:01; HLA-A*33:03; HLA-A*02:06; HLA-A*34:02 and HLA-A*68:02.

[0104] Embodiment 47 is an engineered cell according to any one of Embodiments 3 to 24 and 26 to 45, wherein the engineered cell is homozygous for HLA-A, the engineered cell is homozygous for HLA-C, and the HLA-A allele is one of the following HLA-A alleles: HLA-A*02:01; HLA-A*01:01; HLA-A*03:01; HLA-A*11:01; HLA-A*26:01; HLA-A*68:01; HLA-A*29:02; HLA-A*31:01; HLA-A*32:01; HLA-A*30:02; HLA-A*25:01; HLA-A*33:01; HLA-A*02:02; HLA-A*74:01; HLA-A*02:02; HLA-A*29:01; HLA-A*02:03; HLA-A*02:05; HLA-A*24:07; HLA-A*11:02; HLA-A*36:01; HLA-A*02:22; HLA-A*34:02; HLA-A*01:03; HLA-A*24:02; HLA-A*02:07; HLA-A*23:01; HLA-A*30:01; HLA-A*33:03; HLA-A*02:06; HLA-A*34:02; and HLA-A*68:02; and the HLA-C allele is one of the following HLA-C alleles: HLA-C*07:02; HLA-C*07:01; HLA-C*05:01; HLA-C*04:01; HLA-C*03:04; HLA-C*06:02; HLA-C*08:02; HLA-C*08:01; HLA-C*03:02; HLA-C*16:01; HLA-C*15:02; HLA-C*03:04; HLA-C*12:03; HLA-C*02:10; HLA-C*05:01; HLA-C*12:02; HLA-C*14:02; HLA-C*04:01; HLA-C*03:03; HLA-C*07:04; HLA-C*17:01; HLA-C*01:02 and HLA-C*02:02.

[0105] Embodiment 48 is an engineered cell according to any one of Embodiments 1 to 47, wherein the cell has reduced or eliminated surface expression of MHC class II protein.

[0106] Embodiment 49 is an engineered cell according to any one of Embodiments 1 to 48, and the cell has a genetic modification of a gene selected from CIITA, HLA-DR, HLA-DQ, HLA-DP, RFX5, RFXB / ANK, RFXAP, CREB, NF-YA, NF-YB, and NF-YC.

[0107] Embodiment 50 is an engineered cell according to any one of Embodiments 1 to 49, and the cell includes a genetic modification in the CIITA gene.

[0108] Embodiment 51 is an engineered cell according to any one of Embodiments 1 to 50, and the cell has reduced or eliminated surface expression of the TRAC protein.

[0109] Embodiment 52 is an engineered cell according to any one of Embodiments 1 to 51, and the cell has reduced or eliminated surface expression of the TRBC protein.

[0110] Embodiment 53 is an engineered cell according to any one of Embodiments 1 to 52, and the genetic modification includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides within the genomic locus, or the genetic modification includes at least 5 consecutive nucleotides within the genomic locus.

[0111] Embodiment 54 is an engineered cell according to any one of Embodiments 1 to 53, and the genetic modification includes at least 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic locus.

[0112] Embodiment 55 is an engineered cell according to any one of Embodiments 1 to 54, and the genetic modification includes an indel.

[0113] Embodiment 56 is the engineered cell according to any one of Embodiments 1 to 55, and the gene modification includes at least one substitution from C to T or at least one substitution from A to G within the genomic coordinates.

[0114] Embodiment 57 is a pharmaceutical composition comprising the engineered cell according to any one of Embodiments 1 to 56.

[0115] Embodiment 58 is a cell population comprising the engineered cell according to any one of Embodiments 1 to 57.

[0116] Embodiment 59 is a pharmaceutical composition comprising the cell population according to Embodiment 58.

[0117] Embodiment 60 is the population according to Embodiment 58 or the pharmaceutical composition according to Embodiment 59, and when measured by flow cytometry, at least 65%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the cell population is HLA-A negative or HLA-B negative.

[0118] Embodiment 61 is the population or the pharmaceutical composition according to any one of Embodiments 58 to 60, and when measured by next-generation sequencing (NGS), at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the cell population comprises the gene modification in the HLA-A gene or the gene modification in the HLA-B gene.

[0119] Embodiment 62 is the population or pharmaceutical composition according to any one of Embodiments 58 to 61, and when measured by flow cytometry, the cell population is at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% CIITA-negative.

[0120] Embodiment 63 is the population or pharmaceutical composition according to any one of Embodiments 58 to 62, and when measured by flow cytometry, the cell population is at least 95%, at least 97%, at least 98%, at least 99% or at least 99.5% endogenous TCR protein-negative.

[0121] Embodiment 64 is a method of administering the engineered cell, cell population, pharmaceutical composition according to any one of Embodiments 1 to 63 to a subject in need thereof.

[0122] Embodiment 65 is a method of administering the engineered cell, cell population or pharmaceutical composition according to any one of Embodiments 1 to 63 to a subject as adoptive cell transfer (ACT) therapy.

[0123] Embodiment 66 is a method of treating a disease or disorder, comprising administering the engineered cell, cell population or pharmaceutical composition according to any one of Embodiments 1 to 63 to a subject in need thereof.

[0124] Embodiment 67 is a composition comprising an HLA-B guide RNA, wherein the HLA-B guide RNA is: i. a guide sequence selected from SEQ ID NOs: 165, 166, 177, 13, 74, 1-12, 14-73, 75-91, 101-164, 167-176, 178-185; ii. at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; iii. a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from SEQ ID NOs: 1-91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75% or 70% identical to a sequence selected from SEQ ID NOs: 101-185; iv. a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or v. a guide sequence complementary to at least 17, 18, 19 or 20 consecutive nucleotides of a genomic region listed in Table 2; or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a genomic region listed in Table 3.

[0125] Embodiment 68 is a composition comprising an HLA-B guide RNA and an HLA-A guide RNA, wherein the HLA-B guide RNA is: i. a guide sequence selected from SEQ ID NOs: 165, 166, 177, 13, 74, 1 to 12, 14 to 73, 75 to 91, and 101 to 164, 167 to 176, 178 to 185; ii. at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1 to 91; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101 to 185; iii. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1 to 91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 101 to 185; iv. a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or v. a guide sequence complementary to at least 17, 18, 19, or 20 consecutive nucleotides of the genomic region listed in Table 2, or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of the genomic region listed in Table 3. The HLA-A guide RNA is: i. a guide sequence selected from SEQ ID NOs: 576, 571, 301 to 570, 572 to 575, 577 to 590; or ii. at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301 to 428 and 463 to 511; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 429 to 462 and 512 to 590; or iii. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301 to 428 and 463 to 511; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 512 to 590; or iv. a guide sequence that binds to a target site comprising a genomic region listed in Tables 4 to 7; or v.It includes a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of the genomic regions listed in Tables 4 to 7.

[0126] Embodiment 69 is a method of producing engineered human cells having reduced or eliminated surface expression of HLA-B protein compared to unmodified cells, said cells being homozygous for HLA-A and homozygous for HLA-C, said method comprising contacting said cells with a composition comprising (i) an HLA-B guide RNA and (ii) optionally an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA-binding agent, wherein said HLA-B guide RNA comprises: i. a guide sequence selected from SEQ ID NOs: 165, 166, 177, 13, 74, 1-12, 14-73, 75-91, 101-164, 167-176, 178-185; ii. at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; iii. a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from SEQ ID NOs: 1-91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75% or 70% identical to a sequence selected from SEQ ID NOs: 101-185; iv. a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or v. a guide sequence complementary to at least 17, 18, 19 or 20 consecutive nucleotides of the genomic regions listed in Table 2, or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of the genomic regions listed in Table 3.

[0127] Embodiment 70 is a method for reducing the surface expression of HLA-B protein as compared to unmodified cells, comprising contacting the cells with a composition comprising (i) an HLA-B guide RNA and (ii) optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the HLA-B guide RNA comprises: i. a guide sequence selected from SEQ ID NOs: 165, 166, 177, 13, 74, 1-12, 14-73, 75-91 and 101-164, 167-176, 178-185; ii. at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; iii. a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from SEQ ID NOs: 1-91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75% or 70% identical to a sequence selected from SEQ ID NOs: 101-185; iv. a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or v. a guide sequence complementary to at least 17, 18, 19 or 20 consecutive nucleotides of a genomic region listed in Table 2; or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a genomic region listed in Table 3.

[0128] Embodiment 71 is a method for producing engineered human cells having reduced or eliminated surface expression of HLA-A and HLA-B proteins compared to unmodified cells, wherein the cells are homozygous for HLA-C, and (a) contacting the cells with a first composition comprising an HLA-B guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the HLA-B guide RNA is: i. a guide sequence selected from SEQ ID NOs: 165, 166, 177, 13, 74, 1-12, 14-73, 75-91, and 101-164, 167-176, 178-185; or ii. at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; or iii. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1-91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 101-185; iv. a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or v. a guide sequence complementary to at least 17, 18, 19, or 20 consecutive nucleotides of a genomic region listed in Table 2, or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a genomic region listed in Table 3, contacting the cells with the first composition; and (b) contacting the cells with a second composition comprising an HLA-A guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the HLA-A guide RNA is: i. a guide sequence selected from SEQ ID NOs: 576, 571, 301-570, 572-575, 577-590; or ii.At least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301 to 428 and 463 to 511; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 429 to 462 and 512 to 590; or iii. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301 to 428 and 463 to 511; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 512 to 590; or iv. a guide sequence that binds to a target site comprising a genomic region listed in Tables 4 to 7; or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a genomic region listed in Tables 4 to 7, contacting the second composition with a cell; and comprising.

[0129] Embodiment 72 is a method for reducing the surface expression of HLA-A protein and HLA-B protein in human cells as compared to unmodified cells, comprising: (a) contacting the cells with a first composition comprising an HLA-B guide RNA and optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the HLA-B guide RNA comprises: i. a guide sequence selected from SEQ ID NOs: 165, 166, 177, 13, 74, 1-12, 14-73, 75-91 and 101-164, 167-176, 178-185; ii. at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; iii. a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from SEQ ID NOs: 1-91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75% or 70% identical to a sequence selected from SEQ ID NOs: 101-185; iv. a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or v. a guide sequence complementary to at least 17, 18, 19 or 20 consecutive nucleotides of a genomic region listed in Table 2; or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a genomic region listed in Table 3, and contacting the cells with the first composition; and (b) contacting the cells with a second composition comprising an HLA-A guide RNA and optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the HLA-A guide RNA comprises: i. a guide sequence selected from SEQ ID NOs: 576, 571, 301-570, 572-575, 577-590; or ii. at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301-428 and 463-511; or at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 429-462 and 512-590; or iii.A guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from SEQ ID NOs: 301 to 428 and 463 to 511; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75% or 70% identical to a sequence selected from SEQ ID NOs: 429 to 462 and 512 to 590; or a guide sequence that binds to a target site comprising a genomic region listed in Tables 4 to 7; or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a genomic region listed in Tables 4 to 7, contacting the cell with a second composition comprising;.

[0130] Embodiment 73 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 72, wherein the RNA-guided DNA binding agent or the nucleic acid encoding the RNA-guided DNA binding agent is SpyCas9, and the HLA-B guide RNA is (i) a guide sequence selected from SEQ ID NOs: 13, 74, 1 to 12, 14 to 73, 75 to 91; or (ii) a guide sequence that is at least 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1 to 91; or (iii) a guide sequence that is at least 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1 to 91; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Table 2; or (v) a guide sequence complementary to at least 17, 18, 19 or 20 consecutive nucleotides of a genomic region listed in Table 2; or (vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from SEQ ID NOs: 1 to 91.

[0131] Embodiment 74 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 72, wherein the RNA-guided DNA binding agent or the nucleic acid encoding the RNA-guided DNA binding agent is NmeCas9, and the HLA-B guide RNA is (i) a guide sequence selected from SEQ ID NOs: 165, 166, 177, 101 to 164, 167 to 176, and 178 to 185; or (ii) a guide sequence that is at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101 to 185; or (iii) a guide sequence that is at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101 to 185; or (iv) a guide sequence that binds to a target site containing the genomic region listed in Table 3; or (v) a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of the genomic region listed in Table 3; or (vi) a guide sequence comprising a sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 101 to 185.

[0132] Embodiment 75 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 72, wherein the RNA-guided DNA binding agent or the nucleic acid encoding the RNA-guided DNA binding agent is NmeCas9, and the HLA-B guide RNA is (i) a guide sequence selected from SEQ ID NOs: 165, 166, 163, 164, 169, and 177; or (ii) a guide sequence that is at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 165, 166, 163, 164, and 177; or (iii) a guide sequence comprising a sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 165, 166, 163, 164, and 177.

[0133] Embodiment 76 is the composition or method according to any one of Embodiments 67 to 75, wherein the HLA-B guide RNA or the HLA-A guide RNA contains at least one modification.

[0134] Embodiment 77 is the composition or method according to any one of Embodiments 67 to 76, wherein the HLA-B guide RNA or the HLA-A guide RNA contains at least one modification, and the at least one modification contains a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0135] Embodiment 78 is the composition or method according to any one of Embodiments 67 to 77, wherein the HLA-B guide RNA or the HLA-A guide RNA contains at least one modification including a phosphorothioate (PS) bond between nucleotides.

[0136] Embodiment 79 is the composition or method according to any one of Embodiments 67 to 78, wherein the HLA-B guide RNA or the HLA-A guide RNA contains at least one modification including a 2'-fluoro (2'-F) modified nucleotide.

[0137] Embodiment 80 is the composition or method according to any one of Embodiments 67 to 79, wherein the HLA-B guide RNA or the HLA-A guide RNA contains a modification in one or more of the first 5 nucleotides at the 5' end of the guide RNA.

[0138] Embodiment 81 is the composition or method according to any one of Embodiments 67 to 80, wherein the HLA-B guide RNA or the HLA-A guide RNA contains a modification in one or more of the last 5 nucleotides at the 3' end of the guide RNA.

[0139] Embodiment 82 is the composition or method according to any one of Embodiments 67 to 81, wherein the HLA-B guide RNA or the HLA-A guide RNA comprises at least one modification comprising a PS bond between the first four nucleotides of the guide RNA.

[0140] Embodiment 83 is the composition or method according to any one of Embodiments 67 to 82, wherein the HLA-B guide RNA or the HLA-A guide RNA comprises at least one modification comprising a PS bond between the last four nucleotides of the guide RNA.

[0141] Embodiment 84 is the composition or method according to any one of Embodiments 67 to 83, wherein the HLA-B guide RNA or the HLA-A guide RNA comprises at least one modification comprising a 2'-O-Me modified nucleotide at the first three nucleotides at the 5' end of the guide RNA.

[0142] Embodiment 85 is the composition or method according to any one of Embodiments 67 to 84, wherein the HLA-B guide RNA or the HLA-A guide RNA comprises at least one modification comprising a 2'-O-Me modified nucleotide at the last three nucleotides at the 3' end of the guide RNA.

[0143] Embodiment 86 is the method according to any one of Embodiments 67 to 85, for example, further comprising reducing or eliminating the surface expression of MHC class II protein in the cell as compared to an unmodified cell by contacting the cell with a gene editing system targeting a gene selected from CIITA, HLA-DR, HLA-DQ, HLA-DP, RFX5, RFXB / ANK, RFXAP, CREB, NF-YA, NF-YB, and NF-YC.

[0144] Embodiment 87 is the method according to any one of Embodiments 67 to 86, further comprising contacting the cell with CIITA guide RNA.

[0145] Embodiment 88 is the method according to any one of Embodiments 67 to 87, and further includes reducing or eliminating the surface expression of TCR protein in the cell as compared with the unmodified cell.

[0146] Embodiment 89 is the method according to any one of Embodiments 67 to 88, and further includes contacting the cell with an exogenous nucleic acid.

[0147] Embodiment 90 is the method according to Embodiment 89, and further includes contacting the cell with an exogenous nucleic acid encoding a targeting receptor.

[0148] Embodiment 91 is the method according to Embodiment 89, and further includes contacting the cell with an exogenous nucleic acid encoding a polypeptide secreted by the cell.

[0149] Embodiment 92 is the method according to Embodiment 89, and further includes contacting the cell with a DNA-dependent protein kinase inhibitor (DNAPKi).

[0150] Embodiment 93 is the method according to Embodiment 92, and the DNAPKi is Compound 1.

[0151] Embodiment 94 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 93, and the cell is an allogeneic cell.

[0152] Embodiment 95 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, and the cell is a primary cell.

[0153] Embodiment 96 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, and the cell is a CD4+ T cell.

[0154] Embodiment 97 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a CD8+ T cell.

[0155] Embodiment 98 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a memory T cell.

[0156] Embodiment 99 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a B cell.

[0157] Embodiment 100 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a plasma B cell.

[0158] Embodiment 101 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a memory B cell.

[0159] Embodiment 102 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a natural killer (NK) cell.

[0160] Embodiment 103 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a macrophage.

[0161] Embodiment 104 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a stem cell.

[0162] Embodiment 105 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a pluripotent stem cell (PSC).

[0163] Embodiment 106 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a hematopoietic stem cell (HSC).

[0164] Embodiment 107 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is an induced pluripotent stem cell (iPSC).

[0165] Embodiment 108 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a mesenchymal stem cell (MSC).

[0166] Embodiment 109 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a neural stem cell (NSC).

[0167] Embodiment 110 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a limbal stem cell (LSC).

[0168] Embodiment 111 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a progenitor cell, for example, a vascular endothelial progenitor cell or a neural progenitor cell.

[0169] Embodiment 112 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is a tissue-specific primary cell.

[0170] Embodiment 113 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is selected from chondrocytes, myocytes and keratinocytes.

[0171] Embodiment 114 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is an activated cell.

[0172] Embodiment 115 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 94, wherein the cell is an inactivated cell.

[0173] Embodiment 116 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 115, and includes an exogenous nucleic acid encoding a polypeptide secreted by the cell, or contacting the cell with the exogenous nucleic acid, wherein the secreted polypeptide is an antibody or an antibody fragment.

[0174] Embodiment 117 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 116, and includes an exogenous nucleic acid encoding a polypeptide secreted by the cell, or contacting the cell with the exogenous nucleic acid, wherein the secreted polypeptide is a full-length IgG antibody.

[0175] Embodiment 118 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 116, and includes an exogenous nucleic acid encoding a polypeptide secreted by the cell, or contacting the cell with the exogenous nucleic acid, wherein the secreted polypeptide is a single-chain antibody.

[0176] Embodiment 119 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 118, and includes an exogenous nucleic acid encoding a polypeptide secreted by the cell, or contacting the cell with the exogenous nucleic acid, wherein the secreted polypeptide is a neutralizing antibody.

[0177] Embodiment 120 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 115, and includes an exogenous nucleic acid encoding a polypeptide secreted by the cell, or contacting the cell with the exogenous nucleic acid, wherein the secreted polypeptide is an enzyme.

[0178] Embodiment 121 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 115, and includes an exogenous nucleic acid encoding a polypeptide secreted by the cell, or contacting the cell with the exogenous nucleic acid, wherein the secreted polypeptide is a cytokine.

[0179] Embodiment 122 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 121, and includes an exogenous nucleic acid encoding a polypeptide secreted by the cell, or contacting the cell with the exogenous nucleic acid, wherein the secreted polypeptide is a fusion protein.

[0180] Embodiment 123 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 122, and includes an exogenous nucleic acid encoding a polypeptide secreted by the cell, or contacting the cell with the exogenous nucleic acid, wherein the secreted polypeptide includes a soluble receptor.

[0181] Embodiment 124 is an engineered cell, cell population, composition, pharmaceutical composition, or method according to any one of Embodiments 1 to 115, and includes an exogenous nucleic acid encoding a targeting receptor, or contacting the cell with the exogenous nucleic acid encoding the targeting receptor, wherein the targeting receptor is a T cell receptor (TCR).

[0182] Embodiment 125 is an engineered cell, cell population, composition, pharmaceutical composition, or method according to any one of Embodiments 1 to 115, and includes an exogenous nucleic acid encoding a targeting receptor, or contacting the cell with an exogenous nucleic acid encoding the targeting receptor, wherein the targeting receptor is a genetically modified TCR.

[0183] Embodiment 126 is an engineered cell, cell population, composition, pharmaceutical composition, or method according to any one of Embodiments 1 to 115, and includes an exogenous nucleic acid encoding a targeting receptor, or contacting the cell with an exogenous nucleic acid encoding the targeting receptor, wherein the targeting receptor is a WT1 TCR.

[0184] Embodiment 127 is an engineered cell, cell population, composition, pharmaceutical composition, or method according to any one of Embodiments 1 to 115, and includes an exogenous nucleic acid encoding a targeting receptor, or contacting the cell with an exogenous nucleic acid encoding the targeting receptor, wherein the targeting receptor is a CAR.

[0185] Embodiment 128 is an engineered cell, cell population, composition, pharmaceutical composition, or method according to any one of Embodiments 1 to 115, and includes an exogenous nucleic acid encoding a targeting receptor, or contacting the cell with an exogenous nucleic acid encoding the targeting receptor, wherein the targeting receptor is a universal CAR.

[0186] Embodiment 129 is an engineered cell, cell population, composition, pharmaceutical composition, or method according to any one of Embodiments 1 to 127, and includes an exogenous nucleic acid encoding a targeting receptor, or contacting the cell with an exogenous nucleic acid encoding the targeting receptor, wherein the targeting receptor is an anti-CD30 CAR.

[0187] Embodiment 130 is an engineered cell, cell population, composition, pharmaceutical composition or method according to any one of Embodiments 1 to 129, and includes an exogenous nucleic acid encoding a targeting receptor, or contacting the cell with an exogenous nucleic acid encoding a targeting receptor, wherein the targeting receptor is an a proliferation-inducing ligand (APRIL).

[0188] Embodiment 131 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 129, wherein the cell is engineered with a gene editing system.

[0189] Embodiment 132 is an engineered cell, cell population, pharmaceutical composition or method according to Embodiment 131, wherein the gene editing system includes a transcription activator-like effector nuclease (TALEN).

[0190] Embodiment 133 is an engineered cell, cell population, pharmaceutical composition or method according to Embodiment 131, wherein the gene editing system includes a zinc finger nuclease.

[0191] Embodiment 134 is an engineered cell, cell population, pharmaceutical composition or method according to Embodiment 131, wherein the gene editing system includes an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0192] Embodiment 135 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 134, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, includes a Cas9 protein.

[0193] Embodiment 136 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is S. pyogenes Cas9.

[0194] Embodiment 137 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is N. meningitidis Cas9, optionally Nme2Cas9.

[0195] Embodiment 138 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is S. thermophilus Cas9.

[0196] Embodiment 139 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is S. aureus Cas9.

[0197] Embodiment 140 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is Cpf1 from F. novicida.

[0198] Embodiment 141 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is Cpf1 from Acidaminococcus sp.

[0199] Embodiment 142 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is Cpf1 from Lachnospiraceae bacterium ND2006.

[0200] Embodiment 143 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is Cas12a.

[0201] Embodiment 144 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is CasX.

[0202] Embodiment 145 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is Mad7 nuclease.

[0203] Embodiment 146 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is ARCUS nuclease.

[0204] Embodiment 147 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is an A to G base editor.

[0205] Embodiment 148 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, is a C to T base editor.

[0206] Embodiment 149 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 135, wherein the RNA-guided DNA binding agent, or the RNA-guided DNA binding agent encoded by the nucleic acid, comprises a cytidine deaminase and an RNA-guided nickase.

[0207] Embodiment 150 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 1 to 149, wherein the cell is engineered by a base editing system comprising a C to T base editor or an A to G base editor.

[0208] Embodiment 151 is an engineered cell, cell population, pharmaceutical composition or method according to any one of the immediately preceding embodiments, wherein the base editing system comprises a polypeptide comprising a cytidine deaminase and an RNA-guided nickase, or a nucleic acid encoding the polypeptide.

[0209] Embodiment 152 is an engineered cell, cell population, pharmaceutical composition or method according to Embodiment 149 or 151, wherein the cytidine deaminase comprises APOBEC3A deaminase (A3A).

[0210] Embodiment 153 is an engineered cell, cell population, pharmaceutical composition or method according to Embodiment 151, wherein the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to SEQ ID NO: 811 or 976.

[0211] Embodiment 154 is an engineered cell, cell population, pharmaceutical composition or method according to Embodiment 151, wherein the nucleic acid encoding the polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to SEQ ID NO: 804 or 822.

[0212] Embodiment 155 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 148 to 154, wherein the base editing system further comprises a uracil glycosylase inhibitor (UGI) in a polypeptide different from the polypeptide comprising a cytidine deaminase and an RNA-guided nickase.

[0213] Embodiment 156 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 148 to 152, wherein the polypeptide comprising the cytidine deaminase and the RNA-guided nickase further comprises a uracil glycosylase inhibitor (UGI).

[0214] Embodiment 157 is the engineered cell, cell population, pharmaceutical composition or method according to Embodiment 156, wherein the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to any one of SEQ ID NOs: 977, 978, 979 and 980.

[0215] Embodiment 158 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 157, wherein the HLA-A guide RNA or the HLA-B guide RNA is provided to the cell in a vector.

[0216] Embodiment 159 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 158, wherein the RNA-guided DNA binder is provided to the cell in a vector, optionally in the same vector as the HLA-A guide RNA or the HLA-B guide RNA.

[0217] Embodiment 160 is the engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 87 to 159, wherein the exogenous nucleic acid is provided to the cell in a vector.

[0218] Embodiment 161 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158 to 160, wherein the vector is a viral vector.

[0219] Embodiment 162 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158 to 160, wherein the vector is a non-viral vector.

[0220] Embodiment 163 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158 to 160, wherein the vector is a lentiviral vector.

[0221] Embodiment 164 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158 to 160, wherein the vector is a retroviral vector.

[0222] Embodiment 165 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158 to 160, wherein the vector is AAV.

[0223] Embodiment 166 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 67 to 165, wherein the guide RNA is provided to the cell in lipid nanoparticles (LNP).

[0224] Embodiment 167 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 67 to 166, wherein the guide RNA is provided to the cell as an RNA-guided DNA binder in the same lipid nanoparticles (LNP).

[0225] Embodiment 168 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 87 to 167, wherein the exogenous nucleic acid is provided to the cell in lipid nanoparticles (LNP).

[0226] Embodiment 169 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 87 to 168, wherein the exogenous nucleic acid is integrated into the genome of the cell.

[0227] Embodiment 170 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 87 to 169, wherein the exogenous nucleic acid is integrated into the genome of the cell by homologous recombination (HR).

[0228] Embodiment 171 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 87 to 170, wherein the exogenous nucleic acid is integrated into a safe harbor locus of the genome of the cell.

[0229] Embodiment 172 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 87 to 171, wherein the exogenous nucleic acid is integrated into the genome of the cell by non-homologous end joining (NHEJ).

[0230] Embodiment 173 is an engineered cell, cell population, pharmaceutical composition, composition, or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 3, 13, 18, 32, 36, 39, 48 - 56, 58, 64 - 71, 73 - 74, 80 - 82, 86, and 88 - 91.

[0231] Embodiment 174 is an engineered cell, cell population, pharmaceutical composition, composition, or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 3, 13, 36, 39, 49 - 56, 64 - 71, 74, 80 - 82, 88, and 90 - 91.

[0232] Embodiment 175 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 13, 39, 49, 52, 65, 74, 82 and 91.

[0233] Embodiment 176 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 3, 39 and 49-52.

[0234] Embodiment 177 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 3, 36, 39, 49, 50, 51 and 52.

[0235] Embodiment 178 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 39, 49 and 52.

[0236] Embodiment 179 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 49, 52-54, 55, 56, 64, 65, 67-71, 73-74, 80-82 and 90.

[0237] Embodiment 180 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 49, 51, 74, 81 and 82.

[0238] Embodiment 181 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 101, 103, 106, 107, 114, 117, 118, 125 to 129, 137, 138, 141, 143, 144, 145, 159, 160, 163, 164, 165, 166, 169, 171, 172, 173, 176, 177, 178, 179 and 180.

[0239] Embodiment 182 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 65 and 74.

[0240] Embodiment 183 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 49, 52 to 54, 56, 64 to 65, 67 to 71, 73 to 74, 80 to 82, 88 and 90 to 91.

[0241] Embodiment 184 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 74, 82 and 91.

[0242] Embodiment 185 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 3, 13, 18, 32, 36, 39, 48 to 56, 58, 64 to 71, 73 to 74, 80 to 82, 86 and 88 to 90.

[0243] Embodiment 186 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 3.

[0244] Embodiment 187 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 13.

[0245] Embodiment 188 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 18.

[0246] Embodiment 189 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 32.

[0247] Embodiment 190 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 36.

[0248] Embodiment 191 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 39.

[0249] Embodiment 192 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 48.

[0250] Embodiment 193 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 49.

[0251] Embodiment 194 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 50.

[0252] Embodiment 195 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 51.

[0253] Embodiment 196 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 52.

[0254] Embodiment 197 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 53.

[0255] Embodiment 198 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 54.

[0256] Embodiment 199 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 55.

[0257] Embodiment 200 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 56.

[0258] Embodiment 201 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 58.

[0259] Embodiment 202 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 64.

[0260] Embodiment 203 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 65.

[0261] Embodiment 204 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 66.

[0262] Embodiment 205 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 67.

[0263] Embodiment 206 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 68.

[0264] Embodiment 207 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 69.

[0265] Embodiment 208 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 70.

[0266] Embodiment 209 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 71.

[0267] Embodiment 210 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 73.

[0268] Embodiment 211 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 74.

[0269] Embodiment 212 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 80.

[0270] Embodiment 213 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 81.

[0271] Embodiment 214 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 82.

[0272] Embodiment 215 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 86.

[0273] Embodiment 216 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 88.

[0274] Embodiment 217 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 89.

[0275] Embodiment 218 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 90.

[0276] Embodiment 219 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 91.

[0277] Embodiment 220 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 101, 103, 106, 107, 114, 117, 118, 125 - 129, 133, 137, 138, 141, 143, 144, 145, 159, 160, 163, 164, 165, 166, 169, 171, 172, 173, 176, 177, 178, 179 and 180.

[0278] Embodiment 221 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 101, 103, 106, 117, 118, 125 - 128, 133, 137 - 138, 141, 143 - 144, 159, 163, 164, 165, 166, 169, 171, 173, 177, 178 and 180.

[0279] Embodiment 222 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 101, 106, 114, 117 - 118, 125 - 128, 133, 137 - 138, 141, 143 - 144, 159, 163, 164, 165, 166, 169, 171, 173, 177, 178 and 180.

[0280] Embodiment 223 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 181172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 101, 117-118, 125-128, 137-138, 144, 159, 163, 164, 165, 166, 169, 177, 178 and 180.

[0281] Embodiment 224 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 101, 117, 127, 137-138, 163, 164, 165, 166, 169 and 177.

[0282] Embodiment 225 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence selected from SEQ ID NOs: 101, 103, 106, 107, 117, 125-129, 137, 138, 141, 143, 144, 145, 159, 160, 163, 164, 165, 166, 169, 171, 172, 173, 176, 177, 178, 179 and 180.

[0283] Embodiment 226 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises a guide sequence comprising a sequence selected from SEQ ID NOs: 163-166, 169 and 177.

[0284] Embodiment 227 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 101.

[0285] Embodiment 228 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 103.

[0286] Embodiment 229 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 106.

[0287] Embodiment 230 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 107.

[0288] Embodiment 231 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 117.

[0289] Embodiment 232 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 125.

[0290] Embodiment 233 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 126.

[0291] Embodiment 234 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 127.

[0292] Embodiment 235 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 128.

[0293] Embodiment 236 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA contains SEQ ID NO: 129.

[0294] Embodiment 237 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA contains SEQ ID NO: 137.

[0295] Embodiment 238 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA contains SEQ ID NO: 138.

[0296] Embodiment 239 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA contains SEQ ID NO: 141.

[0297] Embodiment 240 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA contains SEQ ID NO: 143.

[0298] Embodiment 241 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA contains SEQ ID NO: 144.

[0299] Embodiment 242 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA contains SEQ ID NO: 145.

[0300] Embodiment 243 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA contains SEQ ID NO: 159.

[0301] Embodiment 244 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 160.

[0302] Embodiment 245 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 163.

[0303] Embodiment 246 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 164.

[0304] Embodiment 247 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 165.

[0305] Embodiment 248 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 166.

[0306] Embodiment 249 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 169.

[0307] Embodiment 250 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 171.

[0308] Embodiment 251 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 172.

[0309] Embodiment 252 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 173.

[0310] Embodiment 253 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 176.

[0311] Embodiment 254 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 177.

[0312] Embodiment 255 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 178.

[0313] Embodiment 256 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 179.

[0314] Embodiment 257 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 180.

[0315] Embodiment 258 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises any one of the sequences of SEQ ID NOs: 2186 to 2191.

[0316] Embodiment 259 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 2186.

[0317] Embodiment 260 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 2187.

[0318] Embodiment 261 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 2188.

[0319] Embodiment 262 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 2189.

[0320] Embodiment 263 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 2190.

[0321] Embodiment 264 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 67 to 172, wherein the HLA-B guide RNA comprises SEQ ID NO: 2191.

[0322] Embodiment 265 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 313 or 314.

[0323] Embodiment 266 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 314.

[0324] Embodiment 267 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 315.

[0325] Embodiment 268 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 316.

[0326] Embodiment 269 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 317.

[0327] Embodiment 270 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 318.

[0328] Embodiment 271 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 326.

[0329] Embodiment 272 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 337.

[0330] Embodiment 273 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 338.

[0331] Embodiment 274 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 339.

[0332] Embodiment 275 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 341.

[0333] Embodiment 276 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 343.

[0334] Embodiment 277 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 345.

[0335] Embodiment 278 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 264, wherein the HLA-A guide RNA comprises SEQ ID NO: 362.

[0336] Embodiment 279 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 257, wherein the HLA-A guide RNA comprises SEQ ID NO: 576.

[0337] Embodiment 280 is an engineered cell, cell population, pharmaceutical composition or method according to any one of Embodiments 67 to 257, wherein the HLA-A guide RNA comprises SEQ ID NO: 571.

[0338] Embodiment 281 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 1 to 280 for use in expressing a TCR having specificity for a polypeptide expressed by a cancer cell.

[0339] Embodiment 282 is an engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 1 to 281 for use in administering to a subject as adoptive cell transfer (ACT) therapy.

[0340] Embodiment 283 is the engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 1 to 282 for use in treating a subject having cancer.

[0341] Embodiment 284 is the engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 1 to 283 for use in treating a subject having an infectious disease.

[0342] Embodiment 285 is the engineered cell, cell population, pharmaceutical composition, composition or method according to any one of Embodiments 1 to 284 for use in treating a subject having an autoimmune disease.

[0343] Embodiment 286 is: (a) a cell bank comprising the engineered cell according to any one of Embodiments 1 to 56, 73 to 75, 94 to 285, or the engineered cell generated by the method according to any one of Embodiments 69 to 285; and (b) a catalog comprising information recording the HLA-A and HLA-C alleles of the donor cells in the cell bank.

[0344] Embodiment 287 is the cell bank according to Embodiment 286, wherein the cell bank comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 donor cells having a unique combination of HLA-A alleles and HLA-C alleles as compared to other donor cells in the cell bank.

[0345] Embodiment 288 is a method of administering an engineered cell to a recipient subject in need thereof, the method comprising: (a) determining the HLA-A and HLA-C alleles of the recipient subject; (b) selecting an engineered cell or cell population of Embodiments 1-56, 58, 60-63, 73-75, 94-285, or an engineered cell or cell population generated by the method of any one of Embodiments 69-285, wherein the engineered cell comprises at least one of the same HLA-A or HLA-C alleles as the recipient subject; and (c) administering the selected engineered cell to the recipient subject.

[0346] Embodiment 289 is the method according to Embodiment 288, wherein the subject has the HLA-A and HLA-C alleles of the engineered cell.

[0347] Embodiment 290 is an engineered cell, population, composition, pharmaceutical composition or method according to any one of Embodiments 1-285 for use in administering to a subject who is partially matched for adoptive cell transfer (ACT) therapy, wherein the partially matched subject has the HLA-A and HLA-C alleles of the engineered cell or cell population.

[0348] Embodiment 291 is an engineered cell, population, composition, pharmaceutical composition or method according to any one of Embodiments 64-290, wherein the engineered cell or cell population comprises HLA-A and HLA-C alleles shared with the subject.

[0349] Embodiment 292 is an engineered cell, population, composition, pharmaceutical composition or method according to any one of Embodiments 64-290, wherein the HLA-A and HLA-C alleles of the engineered cell or cell population consist of alleles that match one or more of the HLA-A and HLA-C alleles of the subject.

[0350] Embodiment 293 is an engineered cell, population, composition, pharmaceutical composition, or method according to any one of the preceding Embodiments 64 to 290, wherein the HLA-C alleles of the engineered cell or the cell population consist of alleles that are compatible with one or more HLA-C alleles of the subject.

[0351] Embodiment 294 is a cell bank comprising: (a) engineered cells according to any one of Embodiments 1 to 56, 73 to 75, 94 to 285, or engineered cells generated by the method according to any one of Embodiments 69 to 285; and (b) a catalog containing information recording the HLA-C alleles of the donor cells in the cell bank.

[0352] Embodiment 295 is a method of administering engineered cells to a recipient subject in need thereof, the method comprising: (a) determining the HLA-C alleles of the recipient subject; (b) selecting engineered cells or a cell population according to any one of Embodiments 1 to 56, 58, 60 to 63, 73 to 75, 94 to 285, or engineered cells or a cell population generated by the method according to any one of Embodiments 69 to 285, wherein the engineered cells are homozygous for one of the HLA-C alleles of the recipient subject; and (c) administering the selected engineered cells to the recipient subject.

[0353] Embodiment 296 is the method according to Embodiment 295, wherein the subject is homozygous or heterozygous for the HLA-C alleles of the engineered cells.

[0354] Embodiment 297 is an engineered cell, population, composition, pharmaceutical composition, or method according to any one of Embodiments 1 to 285 for use in administering to a subject who is partially matched for adoptive cell transfer (ACT) therapy, wherein the partially matched subject is homozygous or heterozygous for the HLA-C alleles of the engineered cell or the cell population.

[0355] Embodiment 298 is an engineered cell, population, composition, pharmaceutical composition or method according to any one of Embodiments 64 to 297, wherein the engineered cell or the cell population comprises an HLA-C allele shared with the subject.

[0356] Embodiment 299 is an engineered cell, population, composition, pharmaceutical composition or method according to any one of Embodiments 64 to 298, wherein the HLA-C allele of the engineered cell or the cell population consists of alleles that are compatible with one or more HLA-C alleles of the subject.

[0357] Embodiment 300 is an engineered cell, population, composition, pharmaceutical composition or method according to any one of Embodiments 64 to 299, wherein the HLA-C allele of the engineered cell or the cell population consists of alleles that are compatible with one or both HLA-C alleles of the subject.

[0358] A. Definitions Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings.

[0359] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the terms listed before that term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations that include repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, CBBA, CABA, etc., are explicitly included. One of ordinary skill in the art will understand that, unless otherwise apparent from the context, there is usually no limit to the number of items or terms in any combination.

[0360] As used herein, the term "kit" refers to a packaged set of related components (such as one or more polynucleotides or compositions) and one or more related substances (such as delivery devices (e.g., syringes), solvents, solutions, buffers, instructions, or desiccants).

[0361] As used herein, the term "allogeneic" cells refers to cells obtained from a donor subject of the same species as the recipient subject, and there are genetic differences between the donor subject and the recipient subject. For example, the genes at one or more loci are not identical. That is, for example, the cells are allogeneic to the subject receiving administration of the cells. As used herein, cells removed or isolated from a donor and not reintroduced into the original donor are considered allogeneic cells.

[0362] As used herein, "autologous" cells refer to cells collected from the same subject and to be reintroduced with their cellular material at a later time. That is, for example, cells are considered autologous if they are removed from a subject and later reintroduced into the same subject.

[0363] "β2M" or "B2M", as used herein, refers to the nucleic acid or protein sequence of "β-2 microglobulin", and its human gene has an accession number of NC_000015 (range 44711492..44718877) (reference GRCh38.p13). The B2M protein associates as a heterodimer with MHC class I molecules on the surface of nucleated cells and is required for the expression of MHC class I proteins.

[0364] "CIITA" or "CIITA" or "C2TA", as used herein, refers to the nucleic acid or protein sequence of "class II major histocompatibility complex transactivator", and the accession number of the human gene is NC_000016.10 (range 10866208..10941562) (reference GRCh38.p13). The CIITA protein in the nucleus acts as a positive regulator of the transcription of MHC class II genes and is required for the expression of MHC class II proteins.

[0365] As used herein, "MHC", "MHC molecule(s)", "MHC protein" or "MHC complex(es)" refers to major histocompatibility complex molecule(s), including, for example, MHC class I molecules and MHC class II molecules. In humans, MHC molecules are referred to as the "human leukocyte antigen" complex, "HLA molecule" or "HLA protein". The use of the terms "MHC" and "HLA" is not intended to be limiting, and as used herein, the term "MHC" can be used to refer to human MHC molecules, i.e., HLA molecules. Thus, the terms "MHC" and "HLA" are used interchangeably herein.

[0366] As used herein, the term "HLA-A" refers to an MHC class I protein molecule, a heterodimer composed of a heavy chain (encoded by the HLA-A gene) and a light chain (i.e., beta-2 microglobulin), in the context of the HLA-A protein. As used herein, the term "HLA-A" or "HLA-A gene" refers to, in the context of nucleic acids, the gene encoding the heavy chain of the HLA-A protein molecule. The HLA-A gene, also called "HLA class I histocompatibility, A alpha chain", has the human gene accession number NC_000006.12 (29942532..29945870). The HLA-A gene is known to have thousands of different cell-type versions of the HLA-A gene throughout the population (and an individual can receive two different alleles of the HLA-A gene). Public databases regarding HLA-A alleles, including sequence information, can be obtained at IPD-IMGT / HLA: www.ebi.ac.uk / ipd / imgt / hla / . All of the alleles of HLA-A are included in the terms "HLA-A" and "HLA-A gene".

[0367] As used herein, the term "HLA-B" refers to an MHC class I protein molecule, a heterodimer composed of a heavy chain (encoded by the HLA-B gene) and a light chain (i.e., beta-2 microglobulin), in the context of the HLA-B protein. "HLA-B", as used herein, refers to the gene encoding the heavy chain of the HLA-B protein molecule in the context of nucleic acids. HLA-B is also referred to as "HLA class I histocompatibility, B alpha chain", and its human gene has the accession number NC_000006.12 (31353875..31357179). The HLA-B gene is known to have thousands of different cell-type versions of the HLA-B gene throughout the population (and an individual can receive two different alleles of the HLA-A gene). Public databases regarding HLA-B alleles can be accessed at IPD-IMGT / HLA: www.ebi.ac.uk / ipd / imgt / hla / , including sequence information. All alleles of HLA-B are included in the terms "HLA-B" and "HLA-B gene".

[0368] As used herein, "HLA-C" refers to the gene encoding the heavy chain of the HLA-C protein molecule in the context of nucleic acids. HLA-C is also referred to as "HLA class I histocompatibility, C alpha chain", and its human gene has the accession number NC_000006.12 (31268749..31272092).

[0369] As used herein, the term "within a genomic coordinate" includes the boundaries of the specified genomic coordinate range. For example, if chr6:29942854-chr6:29942913 is specified, it includes the coordinates chr6:29942854-chr6:29942913. Throughout this application, the genomic coordinates referred to are based on genomic annotations in the GRCh38 (also referred to as hg38) assembly of the Genome Reference Consortium's human genome, which is available on the website of the National Center for Biotechnology Information. Tools and methods for converting genomic coordinates between one assembly and another are known in the art, and using them, the genomic coordinates shown herein can be converted to corresponding coordinates in another assembly of the human genome, including conversions to previous assemblies made by the same institution or using the same algorithm (e.g., conversion from GRCh38 to GRCh37), and conversions between assemblies made by different institutions or algorithms (e.g., conversion from GRCh38 to NCBI33 made by the International Human Genome Sequencing Consortium). Available methods and tools known in the art include, but are not limited to, the NCBI Genome Remapping Service (available on the website of the National Center for Biotechnology Information), UCSC LiftOver (available on the website of the UCSC Genome Browser), and Assembly Converter (available on the website of Ensembl.org).

[0370] As used herein, the term "homozygosity" refers to having two identical alleles of a particular gene.

[0371] As used herein, an HLA "allele" can refer to a named HLA-A, HLA-B, or HLA-C gene, and the first 4 digits of the name following "HLA-A", "HLA-B" or "HLA-C" (or the first 2 sets of digits separated by a colon, e.g., HLA-A*02:101:01:02N (the first 2 digits are in bold and italic)) are specified. As is known in the art, the first 4 digits (or the first 2 sets of digits separated by a colon) specify the protein of the allele. For example, HLA-A*02:01 and HLA-A*01:02 are distinct HLA-A alleles. Further genotypes of each allele exist, e.g., HLA-A*02:01:02:01. Further genotypes of a particular allele are considered to be the same allele, e.g., HLA-A*02:01:02:01 and HLA-A*02:01 are the same allele. Thus, an HLA allele is homozygous when the alleles are the same (i.e., when the first 4 digits of the alleles are the same or the first 2 sets of digits separated by a colon are the same).

[0372] "Matched" or "matching" refers to alleles shared between a donor and a recipient, e.g., the same allele.

[0373] "Polynucleotide" and "nucleic acid" are used herein to refer to multimeric compounds that include conventional RNA, DNA, hybrid RNA-DNA, and polymers that are analogs thereof, and that have nucleosides or nucleoside analogs with nitrogen-containing heterocyclic bases or base analogs that are covalently bonded together along a backbone. The nucleic acid "backbone" can be composed of various linkages, including one or more of a sugar-phosphate diester linkage, a peptide-nucleic acid bond ("peptide nucleic acid" or PNA, PCT WO 95 / 32305), a phosphorothioate linkage, a methylphosphonate linkage, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or a similar compound with substitution (e.g., 2'-methoxy substitution or 2'-halide substitution). The nitrogen-containing bases are conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, among others), inosine, purine or pyrimidine derivatives (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituents at the 5- or 6-position (e.g., 5-methylcytosine), purine bases with substituents at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4-alkyl-pyrimidine, U.S. Patent No. 5,378,825 and PCT No. WO93 / 13121). For general discussion, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992). Nucleic acids may contain one or more "abasic" residues when the backbone does not contain nitrogen-containing bases at the position(s) of the polymer (U.S. Patent No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases, and linkages, or both conventional components and substitutions (e.g., polymers containing conventional bases with 2'-methoxy linkages, or both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), which are analogs that contain one or more LNA nucleotide monomers together with bicyclic furanose units locked within an RNA-mimicking sugar conformation, enhancing hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA can differ by having different sugar moieties, with RNA having uracil or its analogs and DNA having thymine or its analogs.

[0374] "Guide RNA", "gRNA", and simply "guide" are used interchangeably herein, for example, for the purpose of referring to a guide that directs an RNA-guided DNA binder to a target DNA, and can be a single guide RNA, or a combination of a crRNA and a trRNA (also known as a tracrRNA). Exemplary gRNAs include class II Cas nuclease guide RNAs in modified or unmodified form. The crRNA and trRNA may associate as one RNA molecule (single guide RNA, sgRNA), or as two separate RNA strands (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA may be a native sequence, or a trRNA sequence having modifications or diversity compared to the native sequence.

[0375] As used herein, the term "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to a target sequence to be bound or modified (e.g., cleaved) by an RNA-guided DNA binding agent. The "guide sequence" may also be referred to as a "targeting sequence" or a "spacer sequence". The guide sequence can be, for example, 20 nucleotides in length in the case of Streptococcus pyogenes (i.e., Spy Cas9 (SpCas9)) and related Cas9 homologs / orthologs. Shorter or longer sequences, such as 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length, can also be used as guides. In some embodiments, the target sequence is, for example, within a gene or on a chromosome and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence can be about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100%. In some embodiments, the guide sequence and the target region can be 100% complementary or identical. In other embodiments, the guide sequence and the target region can contain at least one mismatch. For example, the guide sequence and the target sequence can contain 1, 2, 3 or 4 mismatches, and the full length of the target sequence is at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region can contain 1 to 4 mismatches, in which case the guide sequence contains at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region can contain 1, 2, 3 or 4 mismatches when the guide sequence contains 20 nucleotides.

[0376] Thus, for Neisseria meningitides (i.e., Nme Cas9 (NmeCas9)) and related Cas9 homologs / orthologs, the guide sequence may be 19, 20, 21, preferably 22, 23 or 24 nucleotides in length, or may be 20-25 nucleotides in length. In some embodiments, the target sequence is, for example, within a gene or on a chromosome and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence is at least 80%, 85%, preferably 90% or 95%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least 1 mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence. For example, the guide sequence and the target sequence may contain 1-2, preferably 1 or less mismatches, and the full length of the target sequence is 19, 20, 21, 22, preferably 23, or 24 nucleotides or more. In some embodiments, the guide sequence and the target region may contain 1-2 mismatches when the guide sequence contains at least 24 nucleotides or more. In some embodiments, the guide sequence and the target region may contain 1-2 mismatches when the guide sequence contains 24 nucleotides. That is, the guide sequence and the target region may form a double-stranded region having at least 2× base pairs or more. In certain embodiments, the double-stranded region may contain 1-2 mismatches such that the guide strand and the target sequence are not completely complementary. The mismatch position is known in the art, for example, the PAM distal mismatch tends to be more tolerant than the PAM proximal match. The mismatch tolerance at other positions is known in the art (see, for example, Edraki et al., 2019. Mol. Cell, 73:1-13).

[0377] The target sequences of the RNA-guided DNA binding agents include both the positive and negative strands of genomic DNA (i.e., a given sequence and its reverse complement). This is because the nucleic acid substrate of the RNA-guided DNA binding agent is a double-stranded nucleic acid. Thus, when it is said that a guide sequence is "complementary to the target sequence", it should be understood that the guide sequence can direct the guide RNA to bind to the reverse complement of the target sequence. That is, in some embodiments, when a guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to a specific nucleotide of the target sequence (e.g., the target sequence without PAM), except for the substitution of T with U in the guide sequence.

[0378] As used herein, "RNA-guided DNA binding agent" means a polypeptide or polypeptide complex having RNA binding activity and DNA binding activity, or the DNA binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the presence of PAM and the sequence of the guide RNA. Exemplary RNA-guided DNA binding agents include Cas-based / Cas nickases, and inactivated forms thereof ("dCas DNA binding agents"). As used herein, "Cas nuclease" encompasses Cas-based, Cas nickases, and dCas DNA binding agents. The dCas DNA binding agent can be an inactive nuclease containing a non-functional nuclease domain (RuvC or HNH domain). In some embodiments, the Cas-based or Cas nickase includes a dCas DNA binding agent modified to enable DNA cleavage, for example, via fusion with a FokI domain. Cas-based / Cas nickases and dCas DNA binding agents include the Csm complex or Cmr complex of the type III CRISPR system, their Cas10 subunit, Csm1 subunit or Cmr2 subunit, the Cascade complex of the type I CRISPR system, its Cas3 subunit, and class 2 Cas nucleases.

[0379] As used herein, "Class 2 Cas nuclease" is a single-stranded polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases / nickases (e.g., the H840A or D10A variants of Spy Cas9, and the D16A and H588A of Nme Cas9, e.g., Nme2 Cas9) that further have RNA-guided DNA cleavage or nickase activity, and Class 2 dCas DNA binders in which the cleavage / nickase activity is inactivated. Examples of Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A variant, R661A variant, Q695A variant, Q926A variant), HypaCas9 (e.g., N692A variant, M694A variant, Q695A variant, H698A variant), eSPCas9(1.0) (e.g., K810A variant, K1003A variant, R1060A variant), and eSPCas9(1.1) (e.g., K848A variant, K1003A variant, R1060A variant) proteins, and modifications thereof. The Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9 and contains an RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is incorporated herein by reference in its entirety. See, for example, Tables S1 and S3 of the Zetsche reference. See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0380] Some Cas9 orthologs are obtained from N. meningitidis (Esvelt et al., NAT. METHODS, vol. 10, 2013, 1116-1121; Hou et al., PNAS, vol. 110, 2013, pages 15644-15649) (Nme1Cas9, Nme2Cas9, and Nme3Cas9). The Nme2Cas9 ortholog functions efficiently in mammalian cells, recognizes the N4CC PAM, and can be used for in vivo editing with cognate gRNAs (Ran et al., NATURE, vol. 520, 2015, pages 186-191; Kim et al., NAT. COMMUN., vol. 8, 2017, pages 14500). Nme2Cas9 can be specific and selective, for example, it is possible to have less off-target editing (Lee et al., MOL. THER., vol. 24, 2016, pages 645-654; Kim et al., 2017). See also WO / 2020081568, which describes, for example, the Nme2Cas9 D16A nickase (e.g., pages 28 and 42) (the content of which is hereby incorporated by reference in its entirety). Throughout, "NmeCas9" or "Nme Cas9" is a general term encompassing any type of NmeCas9, including Nme1Cas9, Nme2Cas9, and Nme3Cas9.

[0381] As used herein, the term "editor" refers to an agent that includes a polypeptide capable of making modifications within a DNA sequence. In some embodiments, the editor is a nuclease such as a Cas9-based nuclease. In some embodiments, the editor is capable of deaminating a base within a DNA molecule, which may be referred to as a base editor. In some embodiments, the editor is capable of deaminating cytosine (C) in DNA. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to deaminase APOBEC3A (A3A). In some embodiments, the editor comprises a Cas9 nickase fused to deaminase APOBEC3A (A3A). In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase and UGI. In some embodiments, the editor lacks UGI.

[0382] As used herein, "cytidine deaminase" means a polypeptide or polypeptide complex capable of catalyzing the hydrolysis-dependent deamination of cytidine or deoxycytidine, typically resulting in uridine or deoxyuridine. Cytidine deaminases include enzymes of the cytidine deaminase superfamily, particularly enzymes of the APOBEC family (subgroup enzymes APOBEC1, APOBEC2, APOBEC4, and APOBEC3), activation-induced cytidine deaminase (AID or AICDA), as well as CMP deaminase (see, e.g., Conticello et al., Mol. Biol. Evol. 22:367-77, 2005; Conticello, Genome Biol. 9:229, 2008; Muramatsu et al., J. Biol. Chem. 274:18470-6, 1999; Carrington et al., Cells 9:1690 (2020)).

[0383] As used herein, the term "APOBEC3" refers to an APOBEC3 protein such as an APOBEC3 protein expressed by any one of the seven genes (A3A - A3H) of the human APOBEC3 locus. APOBEC3 can have DNA or RNA editing catalytic activity. The amino acid sequence of APOBEC3A has been described (UniPROT accession ID: p31941) and is included herein as SEQ ID NO: 799. In some embodiments, the APOBEC3 protein is a human APOBEC3 protein or a wild - type protein. Variants include proteins having a sequence different from the wild - type APOBEC3 protein by one or several mutations (i.e., substitutions, deletions, insertions) such as one or several point substitutions. For example, a truncated APOBEC3 sequence could be used, for example, by deleting several N - terminal or C - terminal amino acids, preferably 1 - 4 amino acids at the C - terminus of the APOBEC3 sequence. As used herein, the term "variant" refers to allelic variants, splicing variants, and natural or artificial mutants that are homologous to the APOBEC3 reference sequence. The variant is "functional" in that it exhibits DNA editing or RNA editing catalytic activity. In some embodiments, APOBEC3 (such as human APOBEC3A) has the 57th position of the wild - type amino acids (numbered in the wild - type sequence). In some embodiments, APOBEC3 (such as human APOBEC3A) has asparagine at the 57th position of the amino acids (numbered in the wild - type sequence).

[0384] As used herein, "nickase" is an enzyme that makes single-strand breaks (also known as "nicks") in double-stranded DNA, i.e., an enzyme that cleaves one strand of the DNA double helix but not the other. As used herein, "RNA-guided DNA nickase" means a polypeptide or polypeptide complex having DNA nickase activity, wherein the DNA nickase activity is sequence-specific and determined by the sequence of the RNA. Exemplary RNA-guided DNA nickases include Cas nickases. Cas nickases include the Csm or Cmr complex of the type III CRISPR system, their Cas10 subunit, Csm1 subunit or Cmr2 subunit, the Cascade complex of the type I CRISPR system, its Cas3 subunit, and the nickase forms of class 2 Cas nucleases. Class 2 Cas nickases include variants in which only one of the two catalytic domains is inactivated, and these variants have RNA-guided DNA nickase activity. Class 2 Cas nickases include polypeptides in which either the HNH or RuvC catalytic domain is inactivated, e.g., Cas9, e.g., Cas9 (e.g., the H840A, D10A or N863A variants of SpyCas9, or the D16A variant of NmeCas9).Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain or the RuvC or RuvC-like domain of N. meningitidis include Nme2Cas9 D16A (HNH nickase) and Nme2Cas9 H588A (RuvC nickase), Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A variant, R661A variant, Q695A variant, Q926A variant), HypaCas9 (e.g., N692A variant, M694A variant, Q695A variant, H698A variant), eSPCas9(1.0) (e.g., K810A variant, K1003A variant, R1060A variant), and eSPCas9(1.1) (e.g., K848A variant, K1003A variant, R1060A variant) proteins, and modifications thereof. The Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9 and includes an RuvC-like protein domain. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety. See, for example, Tables S1 and S3 of the Zetsche reference. "Cas9" includes S. pyogenes (Spy) Cas9, the Cas9 variants listed herein, and their equivalents. See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015), Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0385] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains derived from at least two different proteins. Since one protein is located in the amino-terminal (N-terminal) portion or carboxy-terminal (C-terminal) protein of the fusion protein, it can form an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein", respectively. Any of the proteins shown herein can be produced by any method known in the art. For example, the proteins shown herein can be produced by expression and purification of recombinant proteins, a method that is particularly suitable for fusion proteins containing peptide linkers. Methods for expression and purification of recombinant proteins are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)) (the entire contents of which are incorporated herein by reference).

[0386] As used herein, the term "linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties. Typically, a linker is located between or sandwiched between two groups, molecules or other moieties and is linked to each by a covalent bond. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein), such as the "XTEN" linker that is 16 amino acid residues or a variant thereof (see, e.g., the Examples, and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 900), SGSETPGTSESA (SEQ ID NO: 901) or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 902).

[0387] As used herein, the term "uracil glycosylase inhibitor" or "UGI" refers to a protein that can inhibit the base excision repair enzyme uracil DNA glycosylase (UDG).

[0388] As used herein, the "open reading frame" or "ORF" of a gene refers to a sequence consisting of a series of codons that specify the amino acid sequence of the protein encoded by that gene. An ORF begins with a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon, e.g., TAA, TAG or TGA in DNA, or UAA, UAG or UGA in RNA.

[0389] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA associated with an RNA-guided DNA binding agent such as a Cas nuclease, e.g., a Cas endonuclease, Cas nickase or dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA directs an RNA-guided DNA binding agent such as Cas9 to a target sequence, the guide RNA hybridizes to the target sequence, the binding agent binds to the target sequence, and if the binding agent is an endonuclease or nickase, it can cleave or nick after binding.

[0390] As used herein, a first sequence is considered to "include a sequence having at least X% identity to a second sequence when alignment of the first and second sequences shows that at least X% of the entire positions of the second sequence match the first sequence." For example, the sequence AAGA includes sequences having 100% identity to the sequence AAG, because alignment results in 100% identity in that there is a match at all three positions of the second sequence. Differences between RNA and DNA (generally an exchange of uridine for thymidine or vice versa), and the presence of nucleoside analogs such as modified uridines, do not contribute to identity or complementarity between polynucleotides as long as the relevant nucleotides (e.g., thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, in the sequence 5'-AXG, where X is any modified uridine, e.g., pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, all are considered 100% identical to AUG because they are all completely complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms well known in the art. One of ordinary skill in the art will understand which algorithm settings and parameter settings are appropriate for a given pair of sequences to be aligned. For sequences predicted to be of approximately the same length and having greater than 50% amino acid identity or greater than 75% nucleotide identity, the Needleman-Wunsch algorithm interface, with the Needleman-Wunsch algorithm (supplied by the EBI web server at www.ebi.ac.uk) having default settings, is generally appropriate.

[0391] "mRNA" is used herein for the purpose of referring to a polynucleotide and includes an open reading frame that can be translated into a polypeptide (i.e., can function as a substrate for translation by ribosomes and aminoacylated tRNAs). The mRNA can include a phosphate-sugar backbone that includes ribose residues or analogs thereof, such as 2'-methoxyribose residues. In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of ribose residues, 2'-methoxyribose residues, or combinations thereof.

[0392] As used herein, "indel" refers to an insertion or deletion mutation consisting of a significant number of nucleotides that are, for example, inserted, deleted, or both inserted and deleted at a double-strand break (DSB) site of a target nucleic acid. As used herein, when indel formation results in an insertion, the insertion is a random insertion at the DSB site and is generally not directed by or based on the template sequence.

[0393] As used herein, "reduction or elimination" of protein expression in a cell refers to the partial or complete loss of expression of that protein as compared to an unmodified cell. In some embodiments, surface expression of a protein on a cell is measured by flow cytometry and has "reduced" or "eliminated" surface expression as compared to an unmodified cell, as demonstrated by a decrease in the fluorescence signal when stained with the same antibody against the protein. A cell having "reduced" or "eliminated" surface expression of a protein as measured by flow cytometry as compared to an unmodified cell may be referred to as "negative" for the expression of that protein, as demonstrated by a fluorescence signal similar to that of a cell stained with an isotype control antibody. "Reduction" or "elimination" of protein expression can be measured by other known techniques in the art, along with appropriate controls known to those of skill in the art.

[0394] As used herein, "knockdown" refers to reducing the expression of a particular gene product (e.g., protein, mRNA, or both) relative to the expression of an unedited target sequence, for example. Protein knockdown can be measured by detecting the total amount of that protein in cells, from a sample such as the relevant tissue, fluid or cell population. Protein knockdown can also be measured by measuring a surrogate, marker or activity of that protein. Methods for measuring mRNA knockdown are known and include analyzing mRNA isolated from the relevant sample. In some embodiments, "knockdown" may also refer to the absence of some expression of a particular gene product, for example, a decrease in the amount of mRNA transcription or a decrease in the amount of protein expression by a cell or cell population (including in vivo populations as found in tissues).

[0395] As used herein, "knockout" (or "KO") refers to the loss of expression from a particular gene or the loss of a particular protein in a cell. Knockout can reduce expression below the detection level of an assay. Knockout can be measured by detecting the total amount of protein in a cell, tissue or cell population, respectively.

[0396] As used herein, "target sequence" or "genomic target sequence" refers to the nucleic acid sequence of a target gene that has complementarity to the guide sequence of a gRNA. Through the interaction between the target sequence and the guide sequence, an RNA-guided DNA binding agent is induced to bind to that target sequence and potentially nick or cleave within that target sequence (depending on the activity of the binding agent).

[0397] As used herein, "treatment" refers to administering or applying any therapeutic agent to a subject for a disease or disorder, including inhibiting the disease, preventing its onset, alleviating one or more of the symptoms of the disease, curing the disease, or preventing one or more of the symptoms of the disease (including recurrence of the symptoms).

[0398] Hereinafter, specific embodiments of the present invention will be referred to in detail, and examples thereof are illustrated in the accompanying drawings. Although the present invention is described with the illustrated embodiments, it will be understood that those embodiments are not intended to limit the present invention thereto. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents, and these may be included in the present invention as defined by the appended claims and the embodiments included.

[0399] Before explaining the teachings of the present invention in detail, it should be understood that since a given composition or process step may vary, the present disclosure is not limited thereto. It should be noted that, as used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates, reference to "a cell" includes a plurality of cells, and the like.

[0400] Numeric ranges include the numbers defining the range. Measured values and measurable values are understood to be approximate values taking into account significant figures and errors associated with the measurement. Also, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" is not intended to be limiting. It should be understood that all of the above general descriptions and modes for carrying out the invention are merely illustrative and explanatory and do not limit the present teachings.

[0401] Unless otherwise noted specifically in this specification, in this specification, embodiments that describe various components as "including" also contemplate "consisting of" or "essentially consisting of" the described components, and embodiments that are described as "consisting of" various components in this specification also contemplate "including" or "essentially consisting of" the described components. Also, in this specification, embodiments that describe as "essentially consisting of" various components also contemplate "consisting of" or "including" the described components (this interchangeability does not apply to the use of these terms in the claims). The term "or" is used in an inclusive sense, unless the context clearly indicates otherwise, that is, it is used equivalently to "and / or".

[0402] The section headings used in this specification are for organization purposes only and should not be construed as limiting the desired subject matter in any way. If any material incorporated by reference conflicts with any term defined in this specification or any other explicit content of this specification, this specification shall prevail. Although the teachings of the present invention are described with various embodiments, the teachings of the present invention are not intended to be limited to those embodiments. On the contrary, as will be apparent to those skilled in the art, the teachings of the present invention include various alternatives, modifications, and equivalents.

[0403] B. Genetically Modified Cells 1. Engineered Human Cell Compositions The present disclosure provides engineered human cell compositions that include gene modification in the HLA-B gene and have reduced or eliminated surface expression of HLA-B protein as compared to unmodified cells, wherein the cells are homozygous for HLA-A and homozygous for HLA-C. Further, the present disclosure provides engineered human cell compositions that include (i) gene modification in the HLA-A gene and (ii) gene modification in the HLA-B gene and have reduced or eliminated surface expression of HLA-A and HLA-B proteins as compared to unmodified cells, wherein the cells are homozygous for HLA-C. In some embodiments, the engineered human cells are allogeneic cells. In some embodiments, the engineered human cells having reduced or eliminated HLA-B expression or HLA-A and HLA-B expression are useful for adoptive cell transfer therapy. In some embodiments, the engineered human cells include additional genetic modifications to the cell's genome (e.g., reduction or elimination of MHC class II protein, or reduction or elimination of endogenous T cell receptor (TCR) protein, or introduction of exogenous nucleic acids for expression) to obtain cells desirable for allogeneic transplantation purposes.

[0404] In some embodiments, the engineered human cells are autologous cell therapy. In some embodiments, the engineered human cells are transferred into a recipient having the same HLA-A allele as the engineered human cells. In some embodiments, the engineered human cells are transferred into a recipient having the same HLA-C allele as the engineered human cells. In some embodiments, the engineered human cells are transferred into a recipient having the same HLA-A and HLA-C alleles as the engineered human cells. Thus, the engineered human cells disclosed herein provide a partial HLA match to the recipient, thereby reducing the risk of a harmful immune response.

[0405] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, and the cells are homozygous for HLA-A and HLA-C.

[0406] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-A and HLA-B proteins compared to unmodified cells, and the cells are homozygous for HLA-C.

[0407] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within a genomic locus selected from (a) chr6:31354480-31357174 or (b) chr6:31357084-31354647, and the cells are homozygous for HLA-A and HLA-C.

[0408] In some embodiments, engineered human cells are provided that include genetic modifications in the HLA-A and HLA-B genes and have reduced or eliminated surface expression of the HLA-A and HLA-B proteins compared to unmodified cells, wherein (i) the genetic modification in the HLA-A gene includes at least one nucleotide within a genomic locus selected from chr6:29942854-chr6:29942913 and chr6:29943518-chr6:29943619, and (ii) the genetic modification in the HLA-B gene includes at least one nucleotide within a genomic locus selected from (a) chr6:31354480-31357174 or (b) chr6:31354623-31357108 or 31354497-31357157, and the cells are homozygous for HLA-C.

[0409] In some embodiments, for each of the predetermined genomic coordinate ranges, the range may include ±10 nucleotides at either end of the designated coordinates. For example, if chr6:29942854-chr6:29942913 is shown, in some embodiments, the genomic target sequence or genetic modification may be included in chr6:29942844-chr6:29942923. In some embodiments, for each of the predetermined genomic coordinate ranges, the range may include ±5 nucleotides at either end of the range.

[0410] In some embodiments, the predetermined genomic coordinate range may include the target sequence on both strands of the DNA (i.e., the plus (+) strand and the minus (-) strand).

[0411] Genetic modification of the HLA-A or HLA-B gene is further described herein. In some embodiments, the genetic modification of the HLA-A or HLA-B gene includes any one or more of insertion, deletion, substitution, or deamination of at least one nucleotide in the target sequence.

[0412] The engineered human cells described herein may include a genetic modification in any HLA-B allele of the HLA-B gene, or a genetic modification in any HLA-A allele of the HLA-A gene. The HLA gene is located on chromosome 6 in a genomic region called the HLA superlocus, and in the art, hundreds of HLA-A and HLA-B alleles have been reported (see, for example, Shiina et al., Journal of Human Genetics 54:15-39 (2009)). The sequences of HLA-A and HLA-B alleles are available in the art (see, for example, the IPD-IMGT / HLA database https: / / www.ebi.ac.uk / ipd / imgt / hla / allele.html for searching for the sequences of specific HLA-A and HLA-B alleles).

[0413] In some embodiments, the cell has reduced or eliminated expression of at least one HLA-A allele selected from HLA-A1, HLA-A2, HLA-A3, HLA-A11, and HLA-A24. In some embodiments, the cell has reduced or eliminated expression of HLA-A1. In some embodiments, the cell has reduced or eliminated expression of HLA-A2. In some embodiments, the cell has reduced or eliminated expression of HLA-A3. In some embodiments, the cell has reduced or eliminated expression of HLA-A11. In some embodiments, the cell has reduced or eliminated expression of HLA-A24.

[0414] In some embodiments, the cell has reduced or eliminated expression of at least one HLA-B allele selected from HLA-B7, HLA-B8, HLA-B13, HLA-B21, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-B41, HLA-B42, HLA-B44, HLA-B45, HLA-B46, HLA-B47, HLA-B48, HLA-B49, HLA-B50, HLA-B51, HLA-B52, HLA-B56, HLA-B67, HLA-B73, HLA-B81 and HLA-B83. In some embodiments, the cell has reduced or eliminated expression of HLA-B7. In some embodiments, the cell has reduced or eliminated expression of HLA-B8. In some embodiments, the cell has reduced or eliminated expression of HLA-B13. In some embodiments, the cell has reduced or eliminated expression of HLA-B21. In some embodiments, the cell has reduced or eliminated expression of HLA-B27. In some embodiments, the cell has reduced or eliminated expression of HLA-B35. In some embodiments, the cell has reduced or eliminated expression of HLA-B37. In some embodiments, the cell has reduced or eliminated expression of HLA-B38. In some embodiments, the cell has reduced or eliminated expression of HLA-B39. In some embodiments, the cell has reduced or eliminated expression of HLA-B40. In some embodiments, the cell has reduced or eliminated expression of HLA-B41. In some embodiments, the cell has reduced or eliminated expression of HLA-B42. In some embodiments, the cell has reduced or eliminated expression of HLA-B44. In some embodiments, the cell has reduced or eliminated expression of HLA-B45. In some embodiments, the cell has reduced or eliminated expression of HLA-B46. In some embodiments, the cell has reduced or eliminated expression of HLA-B47. In some embodiments, the cell has reduced or eliminated expression of HLA-B48.In some embodiments, the cell has a reduced or eliminated expression of HLA - B49. In some embodiments, the cell has a reduced or eliminated expression of HLA - B50. In some embodiments, the cell has a reduced or eliminated expression of HLA - B51. In some embodiments, the cell has a reduced or eliminated expression of HLA - B52. In some embodiments, the cell has a reduced or eliminated expression of HLA - B56. In some embodiments, the cell has a reduced or eliminated expression of HLA - B57. In some embodiments, the cell has a reduced or eliminated expression of HLA - B67. In some embodiments, the cell has a reduced or eliminated expression of HLA - B73. In some embodiments, the cell has a reduced or eliminated expression of HLA - B81. In some embodiments, the cell has a reduced or eliminated expression of HLA - B83.

[0415] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31355182-31355202; chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355347-31355367; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429.

[0416] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31355182-31355202; chr6:31355349-31355369; chr6:31355348-31355368; or chr6:31355145-31355165.

[0417] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, and the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31355348 - 31355368; chr6:31355349 - 31355369; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355182 - 31355202; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355145 - 31355165; chr6:31355432 - 31355452; chr6:31355340 - 31355360; or chr6:31355414 - 31355434.

[0418] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, the genetic modification including at least one nucleotide within genomic coordinates selected from chr6:31355348-31355368; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355182-31355202; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355145-31355165; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429.

[0419] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, the genetic modification including at least one nucleotide within genomic coordinates selected from chr6:31355348-31355368, chr6:31355349-31355369, chr6:31355192-31355212, chr6:31355347-31355367, chr6:31355340-31355360, chr6:31355409-31355429.

[0420] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within a genomic locus selected from chr6:31355349-31355369 or chr6:31355348-31355368.

[0421] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within a genomic locus selected from chr6:31355192-31355212 or chr6:31355347-31355367.

[0422] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within a genomic locus selected from chr6:31355347-31355367; chr6:31355340-31355360; or chr6:31355409-31355429.

[0423] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within a genomic locus selected from chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355347-31355367; chr6:31355432-31355452; or chr6:31355340-31355360.

[0424] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from chr6:31355182-31355202;chr6:31355348-31355368;chr6:31355180-31355200;chr6:31355145-31355165;chr6:31355349-31355369;chr6:31355157-31355177;chr6:31356381-31356401;chr6:31356380-31356400;chr6:31355204-31355224;chr6:31355205-31355225;chr6:31355185-31355205;chr6:31355191-31355211;chr6:31355192-31355212;chr6:31355190-31355210;chr6:31355193-31355213;chr6:31355198-31355218;chr6:31355320-31355340;chr6:31355319-31355339;chr6:31355178-31355198;chr6:31355347-31355367;chr6:31355432-31355452;chr6:31355340-31355360;chr6:31355576-31355596;chr6:31355410-31355430;chr6:31355419-31355439;chr6:31355414-31355434;chr6:31355409-31355429.

[0425] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells, wherein the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425; and chr6:31355469 - 31355493.

[0426] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355361-31355385; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355356-31355380; chr6:31355366-31355390; chr6:31355417-31355441; chr6:31357078-31357102; chr6:31355460-31355484; chr6:31355415-31355439; chr6:31355166-31355190; chr6:31355378-31355402; chr6:31355401-31355425; chr6:31356262-31356286; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; or chr6:31356764-31356788.

[0427] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, the genetic modification including at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; or chr6:31356426-31356450.

[0428] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, the genetic modification including at least one nucleotide within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; or chr6:31355441-31355465.

[0429] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells, and the genetic modification includes at least one nucleotide within genomic coordinates selected from chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; or chr6:31355441 - 31355465.

[0430] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425; chr6:31355469 - 31355493; chr6:31356262 - 31356286; chr6:31355419 - 31355443; chr6:31355390 - 31355414; chr6:31355369 - 31355393; chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; chr6:31355441 - 31355465; chr6:31355203 - 31355227; chr6:31356437 - 31356461; chr6:31356426 - 31356450; chr6:31356763 - 31356787; chr6:31356764 - 31356788; chr6:31356762 - 31356786; chr6:31355204 - 31355228; chr6:31356436 - 31356460; or chr6:31356767 - 31356791.

[0431] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, and the genetic modification includes at least one nucleotide within genomic coordinates selected from (a) chr6:31355348-31355368; or (b) chr6:31355390-31355414; chr6:31355417-31355441; or chr6:31356386-31356410.

[0432] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, and the genetic modification includes indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from (a) chr6:31355145-31356401 or (b) chr6:31357084-31354647. In some embodiments, the cells are homozygous for HLA-A and homozygous for HLA-C.

[0433] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-A and HLA-B genes and have reduced or eliminated surface expression of the HLA-A and HLA-B proteins as compared to unmodified cells, wherein the genetic modification in the HLA-A gene includes indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr6:29942854-chr6:29942913 and chr6:29943518-chr6:29943619, and the genetic modification in the HLA-B gene includes indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from (a) chr6:31355145-31356401 or (b) chr6:31357084-31354647. In some embodiments, the cells are homozygous for HLA-C.

[0434] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, and the genetic modification includes an indel, a C - to - T substitution, or an A - to - G substitution within genomic coordinates selected from chr6:31355182 - 31355202; chr6:31355348 - 31355368; chr6:31355145 - 31355165; chr6:31355349 - 31355369; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355347 - 31355367; chr6:31355432 - 31355452; chr6:31355340 - 31355360; chr6:31355410 - 31355430; chr6:31355414 - 31355434; or chr6:31355409 - 31355429. In some embodiments, the cells are homozygous for HLA - A and HLA - C.

[0435] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, and the genetic modification includes an indel, a C - to - T substitution, or an A - to - G substitution within genomic coordinates selected from chr6:31355182 - 31355202; chr6:31355349 - 31355369; chr6:31355348 - 31355368; or chr6:31355145 - 31355165. In some embodiments, the cells are homozygous for HLA - A and HLA - C.

[0436] In some embodiments, engineered human cells are provided that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, the genetic modification comprising an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31355348-31355368; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355182-31355202; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355145-31355165; chr6:31355432-31355452; chr6:31355340-31355360; or chr6:31355414-31355434. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0437] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, the genetic modification including an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31355348-31355368; chr6:31355349-31355369; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355205-31355225; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355182-31355202; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355145-31355165; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355410-31355430; chr6:31355414-31355434; or chr6:31355409-31355429. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0438] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, the genetic modification including an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31355348-31355368, chr6:31355349-31355369, chr6:31355192-31355212, chr6:31355347-31355367, chr6:31355340-31355360, chr6:31355409-31355429. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0439] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification includes an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31355349-31355369 or chr6:31355348-31355368. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0440] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification includes an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31355192-31355212 or chr6:31355347-31355367. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0441] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification includes an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31355347-31355367; chr6:31355340-31355360; or chr6:31355409-31355429. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0442] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31355348-31355368; chr6:31355145-31355165; chr6:31355347-31355367; chr6:31355432-31355452; or chr6:31355340-31355360. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0443] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355415-31355439; chr6:31355378-31355402; chr6:31355166-31355190; chr6:31355401-31355425; chr6:31355469-31355493; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; or chr6:31356764-31356788. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0444] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, wherein the genetic modification comprises an indel, a C - to - T substitution, or an A - to - G substitution within genomic coordinates selected from chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355361 - 31355385; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355356 - 31355380; chr6:31355366 - 31355390; chr6:31355417 - 31355441; chr6:31357078 - 31357102; chr6:31355460 - 31355484; chr6:31355415 - 31355439; chr6:31355166 - 31355190; chr6:31355378 - 31355402; chr6:31355401 - 31355425; chr6:31356262 - 31356286; chr6:31355221 - 31355245; chr6:31355222 - 31355246; chr6:31355205 - 31355229; chr6:31355446 - 31355470; chr6:31356425 - 31356449; chr6:31355441 - 31355465; chr6:31355203 - 31355227; chr6:31356437 - 31356461; chr6:31356426 - 31356450; chr6:31356763 - 31356787; or chr6:31356764 - 31356788. In some embodiments, the cells are homozygous for HLA - A and HLA - C.

[0445] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, wherein the genetic modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; or chr6:31356426-31356450. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0446] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, and the genetic modification includes an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; or chr6:31355441-31355465. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0447] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, and the genetic modification includes an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; or chr6:31355441-31355465. In some embodiments, the cells are homozygous for HLA-A and HLA-C.

[0448] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of the HLA - B protein compared to unmodified cells, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from (a) chr6:31355348 - 31355368; or (b) chr6:31355390 - 31355414; chr6:31355417 - 31355441; or chr6:31356386 - 31356410. In some embodiments, the cells are homozygous for HLA - A and HLA - C.

[0449] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, and the genetic modification is (a) chr6:31355182 - 31355202; chr6:31355348 - 31355368; chr6:31355180 - 31355200; chr6:31355145 - 31355165; chr6:31355349 - 31355369; chr6:31355157 - 31355177; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355185 - 31355205; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355190 - 31355210; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355432 - 31355452; chr6:31355340 - 31355360; chr6:31355576 - 31355596; chr6:31355410 - 31355430; chr6:31355419 - 31355439; chr6:31355414 - 31355434; chr6:31355409 - 31355429; or (b) chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402;Genomic coordinates selected from chr6:31355166-31355190;chr6:31355401-31355425;ch6:31355469-31355493;chr6:31356262-31356286;chr6:31355419-31355443;chr6:31355390-31355414;chr6:31355369-31355393;chr6:31355221-31355245;chr6:31355222-31355246;chr6:31355205-31355229;chr6:31355446-31355470;chr6:31356425-31356449;chr6:31355441-31355465;chr6:31355203-31355227;chr6:31356437-31356461;chr6:31356426-31356450;chr6:31356763-31356787;chr6:31356764-31356788;chr6:31356762-31356786;chr6:31355204-31355228;chr6:31356436-31356460; or chr6:31356767-31356791, including indels, C to T substitutions or A to G substitutions within the genomic coordinates, and the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides within the genomic coordinates. In some embodiments, the cell is homozygous for HLA-A. In some embodiments, the cell is homozygous for HLA-C. In some embodiments, the cell is homozygous for HLA-A and homozygous for HLA-C.;

[0450] In some embodiments, engineered human cells are provided that include genetic modification in the HLA - B gene and have reduced or eliminated surface expression of HLA - B protein compared to unmodified cells, and the genetic modification is (a) chr6:31355182 - 31355202; chr6:31355348 - 31355368; chr6:31355180 - 31355200; chr6:31355145 - 31355165; chr6:31355349 - 31355369; chr6:31355157 - 31355177; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355185 - 31355205; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355190 - 31355210; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355432 - 31355452; chr6:31355340 - 31355360; chr6:31355576 - 31355596; chr6:31355410 - 31355430; chr6:31355419 - 31355439; chr6:31355414 - 31355434; chr6:31355409 - 31355429; or (b) chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402;Genomic coordinates selected from chr6:31355166-31355190;chr6:31355401-31355425;ch6:31355469-31355493;chr6:31356262-31356286;chr6:31355419-31355443;chr6:31355390-31355414;chr6:31355369-31355393;chr6:31355221-31355245;chr6:31355222-31355246;chr6:31355205-31355229;chr6:31355446-31355470;chr6:31356425-31356449;chr6:31355441-31355465;chr6:31355203-31355227;chr6:31356437-31356461;chr6:31356426-31356450;chr6:31356763-31356787;chr6:31356764-31356788;chr6:31356762-31356786;chr6:31355204-31355228;chr6:31356436-31356460; or chr6:31356767-31356791, including indels, C to T substitutions, or A to G substitutions within the genomic coordinates, wherein the genetic modification comprises at least 5 consecutive nucleotides within the genomic coordinates. In some embodiments, the cell is homozygous for HLA-A. In some embodiments, the cell is homozygous for HLA-C. In some embodiments, the cell is homozygous for HLA-A and homozygous for HLA-C.;

[0451] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, and the genetic modification is (a) chr6:31355182-31355202;chr6:31355348-31355368;chr6:31355180-31355200;chr6:31355145-31355165;chr6:31355349-31355369;chr6:31355157-31355177;chr6:31356381-31356401;chr6:31356380-31356400;chr6:31355204-31355224;chr6:31355205-31355225;chr6:31355185-31355205;chr6:31355191-31355211;chr6:31355192-31355212;chr6:31355190-31355210;chr6:31355193-31355213;chr6:31355198-31355218;chr6:31355320-31355340;chr6:31355319-31355339;chr6:31355178-31355198;chr6:31355347-31355367;chr6:31355432-31355452;chr6:31355340-31355360;chr6:31355576-31355596;chr6:31355410-31355430;chr6:31355419-31355439;chr6:31355414-31355434;chr6:31355409-31355429; or (b) chr6:31356777-31356801;chr6:31355492-31355516;chr6:31355379-31355403;chr6:31355491-31355515;chr6:31355361-31355385;chr6:31355356-31355380;chr6:31355460-31355484;chr6:31357078-31357102;chr6:31355417-31355441;chr6:31355366-31355390;chr6:31355415-31355439;chr6:31355378-31355402;Genomic coordinates selected from chr6:31355166-31355190;chr6:31355401-31355425;ch6:31355469-31355493;chr6:31356262-31356286;chr6:31355419-31355443;chr6:31355390-31355414;chr6:31355369-31355393;chr6:31355221-31355245;chr6:31355222-31355246;chr6:31355205-31355229;chr6:31355446-31355470;chr6:31356425-31356449;chr6:31355441-31355465;chr6:31355203-31355227;chr6:31356437-31356461;chr6:31356426-31356450;chr6:31356763-31356787;chr6:31356764-31356788;chr6:31356762-31356786;chr6:31355204-31355228;chr6:31356436-31356460; or chr6:31356767-31356791, including indels, C to T substitutions, or A to G substitutions, wherein the genetic modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 6 consecutive nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 7 consecutive nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 8 consecutive nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 9 consecutive nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 10 consecutive nucleotides within the genomic coordinates. In some embodiments, the cell is homozygous for HLA-A. In some embodiments, the cell is homozygous for HLA-C. In some embodiments, the cell is homozygous for HLA-A and homozygous for HLA-C.;

[0452] In some embodiments, engineered human cells are provided that include genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein compared to unmodified cells, and the genetic modification is (a) chr6:31355182-31355202; chr6:31355348-31355368; chr6:31355180-31355200; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31355157-31355177; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355185-31355205; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355190-31355210; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355576-31355596; chr6:31355410-31355430; chr6:31355419-31355439; chr6:31355414-31355434; chr6:31355409-31355429; or (b) chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355415-31355439; chr6:31355378-31355402;chr6: 31355166 - 31355190; chr6: 31355401 - 31355425; ch6: 31355469 - 31355493; chr6: 31356262 - 31356286; chr6: 31355419 - 31355443; chr6: 31355390 - 31355414; chr6: 31355369 - 31355393; chr6: 31355221 - 31355245; chr6: 31355222 - 31355246; chr6: 31355205 - 31355229; chr6: 31355446 - 31355470; chr6: 31356425 - 31356449; chr6: 31355441 - 31355465; chr6: 31355203 - 31355227; chr6: 31356437 - 31356461; chr6: 31356426 - 31356450; chr6: 31356763 - 31356787; chr6: 31356764 - 31356788; chr6: 31356762 - 31356786; chr6: 31355204 - 31355228; chr6: 31356436 - 31356460; or an indel, a C to T substitution, or an A to G substitution within a genomic locus selected from chr6: 31356767 - 31356791, wherein the genetic modification comprises at least one C to T substitution or at least one A to G substitution within the genomic locus. In some embodiments, the cell is homozygous for HLA - A. In some embodiments, the cell is homozygous for HLA - C. In some embodiments, the cell is homozygous for HLA - A and homozygous for HLA - C.;

[0453] In some embodiments, an engineered human cell is provided, and gene expression in HLA - B is (a) chr6:31355182 - 31355202; chr6:31355348 - 31355368; chr6:31355180 - 31355200; chr6:31355145 - 31355165; chr6:31355349 - 31355369; chr6:31355157 - 31355177; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355185 - 31355205; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355190 - 31355210; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355432 - 31355452; chr6:31355340 - 31355360; chr6:31355576 - 31355596; chr6:31355410 - 31355430; chr6:31355419 - 31355439; chr6:31355414 - 31355434; and chr6:31355409 - 31355429; or (b) chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425;Reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within a genomic locus selected from ch6:31355469-31355493; chr6:31356262-31356286; chr6:31355419-31355443; chr6:31355390-31355414; chr6:31355369-31355393; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; chr6:31356764-31356788; chr6:31356762-31356786; chr6:31355204-31355228; chr6:31356436-31356460; or chr6:31356767-31356791. In some embodiments, the HLA-B genomic target sequence comprises at least 10 contiguous nucleotides within the genomic locus. In some embodiments, the HLA-B genomic target sequence comprises at least 15 contiguous nucleotides within the genomic locus.;

[0454] In some embodiments, an engineered human cell is provided, and gene expression in HLA - B is (a) chr6:31355182 - 31355202; chr6:31355348 - 31355368; chr6:31355180 - 31355200; chr6:31355145 - 31355165; chr6:31355349 - 31355369; chr6:31355157 - 31355177; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355185 - 31355205; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355190 - 31355210; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355432 - 31355452; chr6:31355340 - 31355360; chr6:31355576 - 31355596; chr6:31355410 - 31355430; chr6:31355419 - 31355439; chr6:31355414 - 31355434; chr6:31355409 - 31355429; or (b) chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425;Reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within a genomic locus selected from ch6:31355469-31355493; chr6:31356262-31356286; chr6:31355419-31355443; chr6:31355390-31355414; chr6:31355369-31355393; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; chr6:31356764-31356788; chr6:31356762-31356786; chr6:31355204-31355228; chr6:31356436-31356460; or chr6:31356767-31356791. In some embodiments, the HLA-B genomic target sequence comprises at least 10 contiguous nucleotides within the genomic locus. In some embodiments, the HLA-B genomic target sequence comprises at least 15 contiguous nucleotides within the genomic locus.;

[0455] In some embodiments, an engineered human cell is provided, and gene expression in HLA - B is (a) chr6:31355182 - 31355202; chr6:31355348 - 31355368; chr6:31355180 - 31355200; chr6:31355145 - 31355165; chr6:31355349 - 31355369; chr6:31355157 - 31355177; chr6:31356381 - 31356401; chr6:31356380 - 31356400; chr6:31355204 - 31355224; chr6:31355205 - 31355225; chr6:31355185 - 31355205; chr6:31355191 - 31355211; chr6:31355192 - 31355212; chr6:31355190 - 31355210; chr6:31355193 - 31355213; chr6:31355198 - 31355218; chr6:31355320 - 31355340; chr6:31355319 - 31355339; chr6:31355178 - 31355198; chr6:31355347 - 31355367; chr6:31355432 - 31355452; chr6:31355340 - 31355360; chr6:31355576 - 31355596; chr6:31355410 - 31355430; chr6:31355419 - 31355439; chr6:31355414 - 31355434; chr6:31355409 - 31355429; or (b) chr6:31356777 - 31356801; chr6:31355492 - 31355516; chr6:31355379 - 31355403; chr6:31355491 - 31355515; chr6:31355361 - 31355385; chr6:31355356 - 31355380; chr6:31355460 - 31355484; chr6:31357078 - 31357102; chr6:31355417 - 31355441; chr6:31355366 - 31355390; chr6:31355415 - 31355439; chr6:31355378 - 31355402; chr6:31355166 - 31355190; chr6:31355401 - 31355425;Reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within a genomic locus selected from ch6:31355469-31355493;chr6:31356262-31356286;chr6:31355419-31355443;chr6:31355390-31355414;chr6:31355369-31355393;chr6:31355221-31355245;chr6:31355222-31355246;chr6:31355205-31355229;chr6:31355446-31355470;chr6:31356425-31356449;chr6:31355441-31355465;chr6:31355203-31355227;chr6:31356437-31356461;chr6:31356426-31356450;chr6:31356763-31356787;chr6:31356764-31356788;chr6:31356762-31356786;chr6:31355204-31355228;chr6:31356436-31356460; or chr6:31356767-31356791. In some embodiments, the HLA-B genomic target sequence comprises at least 10 contiguous nucleotides within the genomic locus. In some embodiments, the HLA-B genomic target sequence comprises at least 15 contiguous nucleotides within the genomic locus.;

[0456] In some embodiments, an engineered human cell is provided, and gene expression in HLA-B is chr6:31355182-31355202;chr6:31355348-31355368;chr6:31355180-31355200;chr6:31355145-31355165;chr6:31355349-31355369;chr6:31355157-31355177;chr6:31356381-31356401;chr6:31356380-31356400;chr6:31355204-31355224;chr6:31355205-31355225;chr6:31355185-31355205;chr6:31355191-31355211;chr6:31355192-31355212;chr6:31355190-31355210;chr6:31355193-31355213;chr6:31355198-31355218;chr6:31355320-31355340;chr6:31355319-31355339;chr6:31355178-31355198;chr6:31355347-31355367;chr6:31355432-31355452;chr6:31355340-31355360;chr6:31355576-31355596;chr6:31355410-31355430;chr6:31355419-31355439;chr6:31355414-31355434;chr6:31355409-31355429; or (b) chr6:31356777-31356801;chr6:31355492-31355516;chr6:31355379-31355403;chr6:31355491-31355515;chr6:31355361-31355385;chr6:31355356-31355380;chr6:31355460-31355484;chr6:31357078-31357102;chr6:31355417-31355441;chr6:31355366-31355390;chr6:31355415-31355439;chr6:31355378-31355402;chr6:31355166-31355190;chr6:31355401-31355425;Reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within a genomic locus selected from ch6:31355469-31355493; chr6:31356262-31356286; chr6:31355419-31355443; chr6:31355390-31355414; chr6:31355369-31355393; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; chr6:31356764-31356788; chr6:31356762-31356786; chr6:31355204-31355228; chr6:31356436-31356460; or chr6:31356767-31356791.;

[0457] In some embodiments, an engineered human cell is provided, and gene expression in HLA-B is determined at chr6:31355182-31355202;chr6:31355348-31355368;chr6:31355180-31355200;chr6:31355145-31355165;chr6:31355349-31355369;chr6:31355157-31355177;chr6:31356381-31356401;chr6:31356380-31356400;chr6:31355204-31355224;chr6:31355205-31355225;chr6:31355185-31355205;chr6:31355191-31355211;chr6:31355192-31355212;chr6:31355190-31355210;chr6:31355193-31355213;chr6:31355198-31355218;chr6:31355320-31355340;chr6:31355319-31355339;chr6:31355178-31355198;chr6:31355347-31355367;chr6:31355432-31355452;chr6:31355340-31355360;chr6:31355576-31355596;chr6:31355410-31355430;chr6:31355419-31355439;chr6:31355414-31355434;chr6:31355409-31355429; or (b) chr6:31356777-31356801;chr6:31355492-31355516;chr6:31355379-31355403;chr6:31355491-31355515;chr6:31355361-31355385;chr6:31355356-31355380;chr6:31355460-31355484;chr6:31357078-31357102;chr6:31355417-31355441;chr6:31355366-31355390;chr6:31355415-31355439;chr6:31355378-31355402;chr6:31355166-31355190;chr6:31355401-31355425;Reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within a genomic locus selected from ch6:31355469-31355493; chr6:31356262-31356286; chr6:31355419-31355443; chr6:31355390-31355414; chr6:31355369-31355393; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; chr6:31356764-31356788; chr6:31356762-31356786; chr6:31355204-31355228; chr6:31356436-31356460; or chr6:31356767-31356791. Due to allelic polymorphisms, in some embodiments, the target sequence may contain 1, 2, or 3 mismatches from the genomic sequence of hg38. In some embodiments, the HLA-B genomic target sequence comprises at least 10 contiguous nucleotides within the genomic locus. In some embodiments, the HLA-B genomic target sequence comprises at least 15 contiguous nucleotides within the genomic locus.;

[0458] In some embodiments, an engineered human cell is provided, and gene expression in HLA-B is determined at chr6:31355182-31355202;chr6:31355348-31355368;chr6:31355180-31355200;chr6:31355145-31355165;chr6:31355349-31355369;chr6:31355157-31355177;chr6:31356381-31356401;chr6:31356380-31356400;chr6:31355204-31355224;chr6:31355205-31355225;chr6:31355185-31355205;chr6:31355191-31355211;chr6:31355192-31355212;chr6:31355190-31355210;chr6:31355193-31355213;chr6:31355198-31355218;chr6:31355320-31355340;chr6:31355319-31355339;chr6:31355178-31355198;chr6:31355347-31355367;chr6:31355432-31355452;chr6:31355340-31355360;chr6:31355576-31355596;chr6:31355410-31355430;chr6:31355419-31355439;chr6:31355414-31355434;chr6:31355409-31355429; or (b) chr6:31356777-31356801;chr6:31355492-31355516;chr6:31355379-31355403;chr6:31355491-31355515;chr6:31355361-31355385;chr6:31355356-31355380;chr6:31355460-31355484;chr6:31357078-31357102;chr6:31355417-31355441;chr6:31355366-31355390;chr6:31355415-31355439;chr6:31355378-31355402;chr6:31355166-31355190;chr6:31355401-31355425;Reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within a genomic locus selected from ch6:31355469-31355493; chr6:31356262-31356286; chr6:31355419-31355443; chr6:31355390-31355414; chr6:31355369-31355393; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; chr6:31356764-31356788; chr6:31356762-31356786; chr6:31355204-31355228; chr6:31356436-31356460; or chr6:31356767-31356791. In some embodiments, the HLA-B genomic target sequence comprises at least 10 contiguous nucleotides within the genomic locus. In some embodiments, the HLA-B genomic target sequence comprises at least 15 contiguous nucleotides within the genomic locus. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent such as a base editor comprising S. pyogenes Cas9, N. meningitidis Cas9, or an S. pyogenes or N. meningitidis Cas9 nickase.;

[0459] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from chr6:31355182-31355202;chr6:31355348-31355368;chr6:31355180-31355200;chr6:31355145-31355165;chr6:31355349-31355369;chr6:31355157-31355177;chr6:31356381-31356401;chr6:31356380-31356400;chr6:31355204-31355224;chr6:31355205-31355225;chr6:31355185-31355205;chr6:31355191-31355211;chr6:31355192-31355212;chr6:31355190-31355210;chr6:31355193-31355213;chr6:31355198-31355218;chr6:31355320-31355340;chr6:31355319-31355339;chr6:31355178-31355198;chr6:31355347-31355367;chr6:31355432-31355452;chr6:31355340-31355360;chr6:31355576-31355596;chr6:31355410-31355430;chr6:31355419-31355439;chr6:31355414-31355434;chr6:31355409-31355429. In some embodiments, the HLA-B genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the HLA-B genomic target sequence comprises at least 15 contiguous nucleotides within the genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent such as S.pyogenes Cas9.

[0460] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least five contiguous nucleotides within genomic coordinates selected from chr6:31356777-31356801; chr6:31355492-31355516; chr6:31355379-31355403; chr6:31355491-31355515; chr6:31355361-31355385; chr6:31355356-31355380; chr6:31355460-31355484; chr6:31357078-31357102; chr6:31355417-31355441; chr6:31355366-31355390; chr6:31355415-31355439; chr6:31355378-31355402; chr6:31355166-31355190; chr6:31355401-31355425; chr6:31355469-31355493; chr6:31356262-31356286; chr6:31355419-31355443; chr6:31355390-31355414; chr6:31355369-31355393; chr6:31355221-31355245; chr6:31355222-31355246; chr6:31355205-31355229; chr6:31355446-31355470; chr6:31356425-31356449; chr6:31355441-31355465; chr6:31355203-31355227; chr6:31356437-31356461; chr6:31356426-31356450; chr6:31356763-31356787; chr6:31356764-31356788; chr6:31356762-31356786; chr6:31355204-31355228; chr6:31356436-31356460; or chr6:31356767-31356791. In some embodiments, the HLA-B genomic target sequence comprises at least ten contiguous nucleotides within the genomic coordinates.In some embodiments, the HLA-B genomic target sequence comprises at least 15 consecutive nucleotides within a genomic locus. In some embodiments, the gene editing system comprises an RNA-guided DNA binder such as N. meningitidis Cas9 or Nme2Cas9.

[0461] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from chr6:31355182-31355202;chr6:31355348-31355368;chr6:31355180-31355200;chr6:31355145-31355165;chr6:31355349-31355369;chr6:31355157-31355177;chr6:31356381-31356401;chr6:31356380-31356400;chr6:31355204-31355224;chr6:31355205-31355225;chr6:31355185-31355205;chr6:31355191-31355211;chr6:31355192-31355212;chr6:31355190-31355210;chr6:31355193-31355213;chr6:31355198-31355218;chr6:31355320-31355340;chr6:31355319-31355339;chr6:31355178-31355198;chr6:31355347-31355367;chr6:31355432-31355452;chr6:31355340-31355360;chr6:31355576-31355596;chr6:31355410-31355430;chr6:31355419-31355439;chr6:31355414-31355434;chr6:31355409-31355429. In some embodiments, the HLA-B genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the HLA-B genomic target sequence comprises at least 15 contiguous nucleotides within the genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent such as a base editor comprising a deaminase and S.pyogenes Cas9 nickase.

[0462] In some embodiments, an engineered human cell is provided, and gene expression in HLA - B is chr6:31355182 - 31355202;chr6:31355348 - 31355368;chr6:31355180 - 31355200;chr6:31355145 - 31355165;chr6:31355349 - 31355369;chr6:31355157 - 31355177;chr6:31356381 - 31356401;chr6:31356380 - 31356400;chr6:31355204 - 31355224;chr6:31355205 - 31355225;chr6:31355185 - 31355205;chr6:31355191 - 31355211;chr6:31355192 - 31355212;chr6:31355190 - 31355210;chr6:31355193 - 31355213;chr6:31355198 - 31355218;chr6:31355320 - 31355340;chr6:31355319 - 31355339;chr6:31355178 - 31355198;chr6:31355347 - 31355367;chr6:31355432 - 31355452;chr6:31355340 - 31355360;chr6:31355576 - 31355596;chr6:31355410 - 31355430;chr6:31355419 - 31355439;chr6:31355414 - 31355434;chr6:31355409 - 31355429; or (b) chr6:31356777 - 31356801;chr6:31355492 - 31355516;chr6:31355379 - 31355403;chr6:31355491 - 31355515;chr6:31355361 - 31355385;chr6:31355356 - 31355380;chr6:31355460 - 31355484;chr6:31357078 - 31357102;chr6:31355417 - 31355441;chr6:31355366 - 31355390;chr6:31355415 - 31355439;chr6:31355378 - 31355402;chr6:31355166 - 31355190;chr6:31355401 - 31355425;Reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within a genomic locus selected from ch6:31355469-31355493;chr6:31356262-31356286;chr6:31355419-31355443;chr6:31355390-31355414;chr6:31355369-31355393;chr6:31355221-31355245;chr6:31355222-31355246;chr6:31355205-31355229;chr6:31355446-31355470;chr6:31356425-31356449;chr6:31355441-31355465;chr6:31355203-31355227;chr6:31356437-31356461;chr6:31356426-31356450;chr6:31356763-31356787;chr6:31356764-31356788;chr6:31356762-31356786;chr6:31355204-31355228;chr6:31356436-31356460; or chr6:31356767-31356791. In some embodiments, the HLA-B genomic target sequence comprises at least 10 consecutive nucleotides within the genomic locus. In some embodiments, the HLA-B genomic target sequence comprises at least 15 consecutive nucleotides within the genomic locus.;

[0463] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within a genomic locus selected from (a) chr6:31355145-31356401 or (b) chr6:31357084-31354647. In some embodiments, the HLA-B genomic target sequence comprises at least 10 consecutive nucleotides within the genomic locus. In some embodiments, the HLA-B genomic target sequence comprises at least 15 consecutive nucleotides within the genomic locus.

[0464] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355182-31355202; chr6:31355348-31355368; chr6:31355180-31355200; chr6:31355145-31355165; chr6:31355349-31355369; chr6:31355157-31355177; chr6:31356381-31356401; chr6:31356380-31356400; chr6:31355204-31355224; chr6:31355205-31355225; chr6:31355185-31355205; chr6:31355191-31355211; chr6:31355192-31355212; chr6:31355190-31355210; chr6:31355193-31355213; chr6:31355198-31355218; chr6:31355320-31355340; chr6:31355319-31355339; chr6:31355178-31355198; chr6:31355347-31355367; chr6:31355432-31355452; chr6:31355340-31355360; chr6:31355576-31355596; chr6:31355410-31355430; chr6:31355419-31355439; chr6:31355414-31355434; and chr6:31355409-31355429.

[0465] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355182-31355202.

[0466] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355348-31355368.

[0467] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355180-31355200.

[0468] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355145-31355165.

[0469] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355349-31355369.

[0470] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355157-31355177.

[0471] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31356381-31356401.

[0472] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31356380-31356400.

[0473] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355204-31355224.

[0474] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355205-31355225.

[0475] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355185-31355205.

[0476] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355191-31355211.

[0477] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355192-31355212.

[0478] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355190-31355210.

[0479] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355193-31355213.

[0480] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355198-31355218.

[0481] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355320-31355340.

[0482] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355319-31355339.

[0483] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355178-31355198.

[0484] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355347-31355367.

[0485] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355432-31355452.

[0486] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355340-31355360.

[0487] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355576-31355596.

[0488] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355410-31355430.

[0489] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355419-31355439.

[0490] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355414-31355434.

[0491] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355409-31355429.

[0492] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31356777-31356801.

[0493] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355492-31355516.

[0494] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355491-31355515.

[0495] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates 31355469-31355493.

[0496] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates 31355460-31355484.

[0497] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates 31355419-31355443.

[0498] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates 31355415-31355439.

[0499] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355417-31355441.

[0500] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355401-31355425.

[0501] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355390-31355414.

[0502] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates 31355379-31355403.

[0503] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates 31355378-31355402.

[0504] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355369-31355393.

[0505] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355361-31355385.

[0506] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355366-31355390.

[0507] In some embodiments, engineered human cells are provided and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355356-31355380.

[0508] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355221-31355245.

[0509] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355222-31355246.

[0510] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355205-31355229.

[0511] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355446-31355470.

[0512] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31356425-31356449.

[0513] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355441-31355465.

[0514] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31355203-31355227.

[0515] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31356437-31356461.

[0516] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31356426-31356450.

[0517] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31356763-31356787.

[0518] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31356764-31356788.

[0519] In some embodiments, engineered human cells are provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates chr6:31356762-31356786.

[0520] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31355204-31355228.

[0521] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31356436-31356460.

[0522] In some embodiments, an engineered human cell is provided, and HLA-B expression is reduced or eliminated by a gene editing system that binds to an HLA-B genomic target sequence comprising at least 5 consecutive nucleotides within genomic coordinates chr6:31356767-31356791.

[0523] In some embodiments, the HLA-B genomic target sequence comprises at least 10 consecutive nucleotides within the genomic coordinates. In some embodiments, the HLA-B genomic target sequence comprises at least 15 consecutive nucleotides within the genomic coordinates.

[0524] In some embodiments, the HLA-B genomic target sequence comprises at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides within the genomic coordinates.

[0525] In some embodiments, the gene editing system comprises a transcription activator-like effector nuclease (TALEN). In some embodiments, the gene editing system comprises a zinc finger nuclease. In some embodiments, the gene editing system comprises a CRISPR / Cas system, such as a class 2 system. In some embodiments, the gene editing system comprises an RNA-guided DNA binder, or a nucleic acid encoding an RNA-guided DNA binder.

[0526] Exemplary RNA-guided DNA binders are shown in Table 1A below. [Table 1]

[0527] In some embodiments, the RNA-guided DNA binder, or the nucleic acid encoding the RNA-guided DNA binder, comprises a Cas9 protein. In some embodiments, the RNA-guided DNA binder is selected from S. pyogenes Cas9, Neisseria meningitidis Cas9, e.g., Nme2Cas9, S. thermophilus Cas9, S. aureus Cas9, Francisella novicida Cpf1, Acidaminococcus sp. Cpf1, Lachnospiraceae bacterium Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, Mad7 nuclease, ARCUS nuclease, and CasX. In some embodiments, the RNA-guided DNA binder comprises a polynucleotide selected from S. pyogenes Cas9, Neisseria meningitidis Cas9, e.g., Nme2Cas9, S. thermophilus Cas9, S. aureus Cas9, Francisella novicida Cpf1, Acidaminococcus sp. Cpf1, Lachnospiraceae bacterium Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, and CasX.

[0528] In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is S. pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is N. meningitidis Cas9, e.g., Nme2Cas9. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is S. thermophilus Cas9. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is S. aureus Cas9. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is Cpf1 from F. novicida. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is Cpf1 from Acidaminococcus sp. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is Cpf1 from Lachnospiraceae bacterium ND2006. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is a C to T base editor. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is an A to G base editor. In some embodiments, the base editor comprises a deaminase and an RNA-guided nickase. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the RNA-guided nickase is SpyCas9 nickase. In some embodiments, the RNA-guided nickase comprises NmeCas9 nickase. In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is Cas12a.In some embodiments, the RNA-guided DNA binding agent, or the nucleic acid encoding the RNA-guided DNA binding agent, is CasX.

[0529] In some of the above embodiments, C comprises an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises Cas9. In some embodiments, the RNA-guided DNA binding agent is S.pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent is a base editor. In some embodiments, the base editor comprises a C to T deaminase and an RNA-guided nickase such as S.pyogenes Cas9 nickase. In some embodiments, the base editor comprises an A to G deaminase and an RNA-guided nickase such as S.pyogenes Cas9 nickase.

[0530] In some of the above embodiments, the gene editing system comprises an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises Cas9. In some embodiments, the RNA-guided DNA binding agent is N.meningitidis or Nme2 Cas9. In some embodiments, the RNA-guided DNA binding agent is a base editor. In some embodiments, the base editor comprises a C to T deaminase and an RNA-guided nickase such as N.meningitidis or Nme2 Cas9 nickase. In some embodiments, the base editor comprises an A to G deaminase and an RNA-guided nickase such as N. meningitidis or Nme2 Cas9 nickase.

[0531] In some embodiments, the gene editing system further comprises a uracil glycosylase inhibitor (UGI), and the UGI and the base editor are included in a single polypeptide. In some embodiments, the gene editing system comprises UGI, and the UGI and the base editor are included in different polypeptides. In some embodiments, the base editor comprises a cytidine deaminase and an RNA-guided nickase. In some embodiments, the cytidine deaminase, the RNA-guided nickase, and the UGI are included in a single polypeptide. In some embodiments, the cytidine deaminase, the RNA-guided nickase, and the UGI are included in different polypeptides. In some embodiments, the cytidine deaminase and the RNA-guided nickase are included in a single polypeptide, and the UGI is included in a different polypeptide.

[0532] In some embodiments, when the engineered cell is homozygous for HLA-A, the HLA-A allele is selected from any one of the following HLA-A alleles: HLA-A*02:01; HLA-A*01:01; HLA-A*03:01; HLA-A*11:01; HLA-A*26:01; HLA-A*68:01; HLA-A*29:02; HLA-A*31:01; HLA-A*32:01; HLA-A*30:02; HLA-A*25:01; HLA-A*33:01; HLA-A*02:02; HLA-A*74:01; HLA-A*02:02; HLA-A*29:01; HLA-A*02:03; HLA-A*02:05; HLA-A*24:07; HLA-A*11:02; HLA-A*36:01; HLA-A*02:22; HLA-A*34:02; HLA-A*01:03; HLA-A*24:02; HLA-A*02:07; HLA-A*23:01; HLA-A*30:01; HLA-A*33:03; HLA-A*02:06; HLA-A*34:02 and HLA-A*68:02.

[0533] In some embodiments, when the engineered cell is homozygous for HLA-C, the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C*07:02; HLA-C*07:01; HLA-C*05:01; HLA-C*04:01; HLA-C*03:04; HLA-C*06:02; HLA-C*08:02; HLA-C*08:01; HLA-C*03:02; HLA-C*16:01; HLA-C*15:02; HLA-C*03:04; HLA-C*12:03; HLA-C*02:10; HLA-C*05:01; HLA-C*12:02; HLA-C*14:02; HLA-C*04:01; HLA-C*03:03; HLA-C*07:04; HLA-C*17:01; HLA-C*01:02; and HLA-C*02:02.

[0534] In some embodiments, when the engineered cell is homozygous for HLA-C, the HLA-C allele is HLA-C*03:04. In some embodiments, when the engineered cell is homozygous for HLA-C, the HLA-C allele is HLA-C*06:02. In some embodiments, when the engineered cell is homozygous for HLA-C, the HLA-C allele is HLA-C*01:02. In some embodiments, when the engineered cell is homozygous for HLA-C, the HLA-C allele is HLA-C*08:01. In some embodiments, when the engineered cell is homozygous for HLA-C, the HLA-C allele is HLA-C*03:02.

[0535] In some embodiments, the engineered cells are homozygous for HLA-A, homozygous for HLA-C, and the HLA-B alleles and HLA-C alleles are selected from the following: HLA-A*01:01 and HLA-C*07:01; HLA-A*02:01 and HLA-C*07:02; HLA-A*02:01 and HLA-C*05:01; HLA-A*03:01 and HLA-C*07:02; HLA-A*02:01 and HLA-C*04:01; HLA-A*02:01 and HLA-C*03:04; HLA-A*01:01 and HLA-C*06:02; HLA-A*03:01 and HLA-C*04:01; HLA-A*02:01 and HLA-C*07:01; HLA-A*24:02 and HLA-C*04:01; HLA-A*29:02 and HLA-C*16:01; HLA-A*02:01 and HLA-C*06:02; HLA-A*24:02 and HLA-C*07:02; HLA-A*26:01 and HLA-C*12:03; HLA-A*11:01 and HLA-C*04:01; HLA-A*25:01 and HLA-C*12:03; HLA-A*02:01 and HLA-C*02:02; HLA-A*24:02 and HLA-C*03:03; HLA-A*30:01 and HLA-C*06:02; HLA-A*02:01 and HLA-C*01:02; HLA-A*11:01 and HLA-C*07:02; HLA-A*03:01 and HLA-C*07:01; HLA-A*23:01 and HLA-C*04:01; HLA-A*24:02 and HLA-C*07:01; HLA-A*31:01 and HLA-C*03:04; HLA-A*33:01 and HLA-C*08:02; HLA-A*02:01 and HLA-C*03:03; HLA-A*11:01 and HLA-C*01:02; HLA-A*01:01 and HLA-C*04:01; HLA-A*03:01 and HLA-C*06:02.

[0536] The HLA-A and HLA-C allele pairs disclosed herein cumulatively cover approximately 81% of the population. The cumulative frequencies of the HLA-A and HLA-C allele pairs are shown in Table 1B below.

Table 2-1

Table 2-2

[0537] In some embodiments, engineered human cells are provided that have reduced or eliminated surface expression of HLA-B protein, compared to unmodified cells, and are homozygous for HLA-A, homozygous for HLA-C, and further comprise reduced or eliminated surface expression of MHC class II proteins. In some embodiments, the engineered human cells comprise in the gene a genetic modification that reduces or eliminates the expression of MHC class II proteins. In some embodiments, the engineered human cells comprise in the CIITA gene a genetic modification. In some embodiments, the engineered human cells comprise in the HLA-DR gene a genetic modification. In some embodiments, the engineered human cells comprise in the HLA-DQ gene a genetic modification. In some embodiments, the engineered human cells comprise in the HLA-DP gene a genetic modification. In some embodiments, the engineered human cells comprise in the RFX gene a genetic modification. In some embodiments, the engineered human cells comprise in the CREB gene a genetic modification. In some embodiments, the engineered human cells comprise in the nuclear factor (NF)-γ gene a genetic modification.

[0538] In some embodiments, engineered human cells are provided that have reduced or eliminated surface expression of HLA-A and HLA-B proteins compared to unmodified cells, which are homozygous for HLA-C and further include reduced or eliminated surface expression of MHC class II proteins. In some embodiments, the engineered human cells include a genetic modification in a gene that reduces or eliminates the expression of MHC class II proteins. In some embodiments, the engineered human cells include a genetic modification in the CIITA gene. In some embodiments, the engineered human cells include a genetic modification in the HLA-DR gene. In some embodiments, the engineered human cells include a genetic modification in the HLA-DQ gene. In some embodiments, the engineered human cells include a genetic modification in the HLA-DP gene. In some embodiments, the engineered human cells include a genetic modification in the RFX gene. In some embodiments, the engineered human cells include a genetic modification in the CREB gene. In some embodiments, the engineered human cells include a genetic modification in the nuclear factor (NF)-γ gene.

[0539] In some embodiments, engineered human cells are provided that have reduced or eliminated surface expression of HLA-B protein compared to unmodified cells, which are homozygous for HLA-A and homozygous for HLA-C and further include reduced or eliminated surface expression of the TRAC protein. In some embodiments, engineered human cells are provided that have reduced or eliminated surface expression of HLA-B protein compared to unmodified cells, which are homozygous for HLA-A and HLA-C and further include reduced or eliminated surface expression of the TRBC protein.

[0540] In some embodiments, engineered human cells are provided that have reduced or eliminated surface expression of HLA-B protein, which are homozygous for HLA-A, homozygous for HLA-C, and further comprise reduced or eliminated surface expression of the TRAC protein. In some embodiments, engineered human cells are provided that have reduced or eliminated surface expression of HLA-B protein compared to unmodified cells, which are homozygous for HLA-C and further comprise reduced or eliminated surface expression of the TRBC protein.

[0541] In some embodiments, engineered human cells are provided that have reduced or eliminated surface expression of HLA-B protein, which are homozygous for HLA-C, and further comprise reduced or eliminated surface expression of the TRAC protein. In some embodiments, engineered human cells are provided that have reduced or eliminated surface expression of HLA-B protein compared to unmodified cells, which are homozygous for HLA-C and further comprise reduced or eliminated surface expression of the TRBC protein.

[0542] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, the genetic modification includes at least one nucleotide within a genomic locus selected from (a) chr6:31355145-31356401 or (b) chr6:31357084-31354647, and the engineered cells further include in the gene a genetic modification that reduces or eliminates the expression of MHC class II proteins. In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, the genetic modification includes at least one nucleotide within a genomic locus selected from (a) chr6:31355182-31355596 or (b) chr6:31355203-31356461, and the engineered cells further include the genetic modification in the CIITA gene.

[0543] In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, the genetic modification includes at least one nucleotide within a genomic locus selected from (a) chr6:31355182-31355596 or (b) chr6:31355203-31356461, and the engineered cells further include the genetic modification in the TRAC gene. In some embodiments, engineered human cells are provided that include a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of the HLA-B protein as compared to unmodified cells, the genetic modification includes at least one nucleotide within a genomic locus selected from (a) chr6:31355182-31355596 or (b) chr6:chr6:31355203-31356461, and the engineered cells further include the genetic modification in the TRBC gene.

[0544] In some embodiments, there are provided engineered human cells that contain a genetic modification in the HLA-B gene and have reduced or eliminated surface expression of HLA-A B protein as compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within a genomic locus selected from (a) chr6:31354480-31357174; chr631355145-31356401 or (b) chr6:chr6:31355203-31356461, and the engineered cells further contain the genetic modification in an exogenous nucleic acid. In some embodiments, the engineered cells contain an exogenous nucleic acid encoding a targeting receptor that is expressed on the surface of the engineered cells. In some embodiments, the targeting receptor is a CAR or a universal CAR. In some embodiments, the targeting receptor is a TCR. In some embodiments, the targeting receptor is a WT1 TCR. In some embodiments, the targeting receptor is a ligand of the receptor. In some embodiments, the targeting receptor is a hybrid CAR / TCR. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain) and a subunit of a TCR. In some embodiments, the targeting receptor is a cytokine receptor. In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a B cell receptor (BCR). In some embodiments, the engineered cells further contain an exogenous nucleic acid encoding a polypeptide secreted by the engineered cells (i.e., a soluble polypeptide). In some embodiments, the exogenous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the secreted polypeptide is an antibody. In some embodiments, the secreted polypeptide is an enzyme. In some embodiments, the exogenous nucleic acid encodes an antibody that encodes a cytokine. In some embodiments, the exogenous nucleic acid encodes a chemokine. In some embodiments, the exogenous nucleic acid encodes a fusion protein.

[0545] The engineered human cell may be any of the exemplary cell types disclosed herein. Additionally, since MHC class I molecules are expressed in all nucleated cells, the engineered human cell can be any nucleated cell. In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is a stem cell such as a hematopoietic stem cell (HSC). In some embodiments, the engineered cell is an induced pluripotent stem cell (iPSC). In some embodiments, the engineered cell is a mesenchymal stem cell (MSC). In some embodiments, the engineered cell is a neural stem cell (NSC). In some embodiments, the engineered cell is a limbal stem cell (LSC). In some embodiments, the engineered cell is a progenitor cell, e.g., a vascular endothelial progenitor cell or a neural progenitor cell. In some embodiments, the engineered cell is a tissue-specific primary cell. In some embodiments, the engineered cell is selected from chondrocytes, myocytes, and keratinocytes. In some embodiments, the engineered cell is a monocyte, macrophage, mast cell, dendritic cell, or granulocyte. In some embodiments, the engineered cell is a monocyte. In some embodiments, the engineered cell is a macrophage. In some embodiments, the engineered cell is a mast cell. In some embodiments, the engineered cell is a dendritic cell. In some embodiments, the engineered cell is a granulocyte. In some embodiments, the engineered cell is a lymphocyte. In some embodiments, the engineered cell is a T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a memory T cell. In some embodiments, the engineered cell is a B cell. In some embodiments, the engineered cell is a plasma B cell. In some embodiments, the engineered cell is a memory B cell. In some embodiments, the engineered cell is a macrophage.

[0546] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one of the engineered human cells disclosed herein. In some embodiments, the pharmaceutical composition comprises a population of any one of the engineered cells disclosed herein. In some embodiments, the population of engineered cells is at least 65% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 70% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 80% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 90% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 91% negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 92% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 93% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 94% HLA-B negative when measured by flow cytometry.

[0547] In some embodiments, the population of engineered cells is at least 94% HLA-A negative or at least 94% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 95% HLA-A negative or at least 95% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 96% HLA-A negative or at least 96% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 97% HLA-A negative or at least 97% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 98% HLA-A negative or at least 98% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 99% HLA-A negative or at least 98% HLA-B negative when measured by flow cytometry.

[0548] In some embodiments, the population of engineered cells is at least 94% HLA-A negative and at least 94% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 95% HLA-A negative and at least 95% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 96% HLA-A negative and at least 96% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 97% HLA-A negative and at least 97% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 98% HLA-A negative and at least 98% HLA-B negative when measured by flow cytometry. In some embodiments, the population of engineered cells is at least 99% HLA-A negative and at least 98% HLA-B negative when measured by flow cytometry.

[0549] In some embodiments, when measured by next-generation sequencing (NGS), at least 92% of the cell population comprises a genetic modification of the HLA-A gene or 92% of the cell population comprises a genetic modification of the HLA-B gene. In some embodiments, the population of engineered cells is at least 93% HLA-A negative or at least 93% HLA-B negative when measured by flow cytometry. In some embodiments, when measured by next-generation sequencing (NGS), at least 93% of the cell population comprises a genetic modification of the HLA-A gene or at least 93% of the cell population comprises a genetic modification of the HLA-B gene. In some embodiments, when measured by next-generation sequencing (NGS), at least 94% of the cell population comprises a genetic modification of the HLA-A gene or at least 94% of the cell population comprises a genetic modification of the HLA-B gene. In some embodiments, when measured by next-generation sequencing (NGS), at least 95% of the cell population comprises a genetic modification of the HLA-A gene or at least 95% of the cell population comprises a genetic modification of the HLA-B gene. In some embodiments, when measured by next-generation sequencing (NGS), at least 96% of the cell population comprises a genetic modification of the HLA-A gene or at least 96% of the cell population comprises a genetic modification of the HLA-B gene. In some embodiments, when measured by next-generation sequencing (NGS), at least 96% of the cell population comprises a genetic modification of the HLA-A gene or at least 97% of the cell population comprises a genetic modification of the HLA-B gene. In some embodiments, when measured by next-generation sequencing (NGS), at least 96% of the cell population comprises a genetic modification of the HLA-A gene or at least 98% of the cell population comprises a genetic modification of the HLA-B gene. In some embodiments, when measured by next-generation sequencing (NGS), at least 96% of the cell population comprises a genetic modification of the HLA-A gene or at least 99% of the cell population comprises a genetic modification of the HLA-B gene.

[0550] In some embodiments, the population of engineered cells is at least 95% CIITA negative as measured by flow cytometry. In some embodiments, the population of engineered cells is at least 96% CIITA negative as measured by flow cytometry. In some embodiments, the population of engineered cells is at least 97% CIITA negative as measured by flow cytometry. In some embodiments, the population of engineered cells is at least 98% CIITA negative as measured by flow cytometry. In some embodiments, the population of engineered cells is at least 99% CIITA negative as measured by flow cytometry.

[0551] In some embodiments, the population of engineered cells is at least 95% negative for endogenous TCR protein as measured by flow cytometry. In some embodiments, the population of engineered cells is at least 97% negative for endogenous TCR protein as measured by flow cytometry. In some embodiments, the population of engineered cells is at least 98% negative for endogenous TCR protein as measured by flow cytometry. In some embodiments, the population of engineered cells is at least 99% negative for endogenous TCR protein as measured by flow cytometry. In some embodiments, the population of engineered cells is at least 99.5% negative for endogenous TCR protein as measured by flow cytometry.

[0552] In some embodiments, provided are methods for administering an engineered human cell or a pharmaceutical composition disclosed herein to a subject in need thereof. In some embodiments, provided are methods for administering an engineered human cell or a pharmaceutical composition disclosed herein to a subject as an ACT therapy. In some embodiments, provided are methods for administering an engineered human cell or a pharmaceutical composition disclosed herein to a subject for the treatment of cancer. In some embodiments, provided are methods for administering an engineered human cell or a pharmaceutical composition disclosed herein to a subject for the treatment of an autoimmune disease. In some embodiments, provided are methods for administering an engineered human cell or a pharmaceutical composition disclosed herein to a subject for the treatment of an infectious disease.

[0553] Methods and compositions for reducing or eliminating surface expression of C.HLA-B The present disclosure provides methods and compositions for reducing or eliminating surface expression of HLA-B protein compared to unmodified cells by genetically modifying the HLA-B gene. The present disclosure provides methods and compositions for reducing or eliminating surface expression of both HLA-A and HLA-B proteins compared to unmodified cells by genetically modifying the HLA-A and HLA-B genes. The resulting genetically modified cells are sometimes referred to herein as engineered cells. In some embodiments, cells that have already been genetically modified (i.e., engineered cells) may be starting cells for further genetic modification using the methods or compositions provided by the present invention. In some embodiments, the cells are allogeneic cells. In some embodiments, cells having reduced or eliminated surface expression of only HLA-B protein, or both HLA-A and HLA-B proteins, are useful in adoptive cell transfer therapy. In some embodiments, editing of the HLA-A or HLA-B gene is combined with additional genetic modification to obtain cells desirable for the purpose of allogeneic transplantation.

[0554] In some embodiments, the method provides a method for reducing the surface expression of HLA-B protein in human cells as compared to unmodified cells, the cell being contacted with a composition comprising (a) a guide RNA comprising (i) a guide sequence selected from SEQ ID NOs: 1-91 and 101-185; or (ii) at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1-91; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 101-185; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or (v) a guide sequence complementary to at least 17, 18, 19, or 20 consecutive nucleotides of a genomic region listed in Table 2, or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a genomic region listed in Table 3; or (vi) a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from (v); and optionally (b) an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0555] In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein. In some embodiments, the RNA-guided DNA binding agent is selected from one of S. pyogenes Cas9, Neisseria meningitidis Cas9, such as Nme2Cas9, S. thermophilus Cas9, S. aureus Cas9, Francisella novicida Cpf1, Acidaminococcus sp. Cpf1, Lachnospiraceae bacterium Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, and CasX. In some embodiments, the RNA-guided DNA binding agent comprises a polynucleotide selected from one of S. pyogenes Cas9, Neisseria meningitidis Cas9, such as Nme2Cas9, S. thermophilus Cas9, S. aureus Cas9, Francisella novicida Cpf1, Acidaminococcus sp. Cpf1, Lachnospiraceae bacterium Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, and CasX. In some embodiments, the RNA-guided DNA binding agent is S. pyogenes Cas9. In some embodiments, the CIITA guide RNA is an S. pyogenes Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9, such as Nme2Cas9. In some embodiments, the RNA-guided DNA binding agent is S. thermophilus Cas9. In some embodiments, the RNA-guided DNA binding agent is S. aureus Cas9.In some embodiments, the RNA-guided DNA binding agent is Cpf1 from F. novicida. In some embodiments, the RNA-guided DNA binding agent is Cpf1 from Acidaminococcus sp. In some embodiments, the RNA-guided DNA binding agent is Cpf1 from Lachnospiraceae bacterium ND2006. In some embodiments, the RNA-guided DNA binding agent is a C to T base editor. In some embodiments, the RNA-guided DNA binding agent is an A to G base editor. In some embodiments, the base editor comprises a deaminase and an RNA-guided nickase. In some embodiments, the RNA-guided DNA binding agent comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the RNA-guided nickase is SpyCas9 nickase. In some embodiments, the RNA-guided nickase comprises NmeCas9 nickase. In some embodiments, the RNA-guided DNA binding agent is Cas12a. In some embodiments, the RNA-guided DNA binding agent is CasX. In some embodiments, the surface expression of the HLA-A protein (i.e., the engineered cell) is thereby reduced or eliminated.

[0556] In some embodiments, the method includes a method of reducing the surface expression of HLA-A and HLA-B proteins in human cells as compared to unmodified cells, the cell being contacted with a first composition comprising: (a) an HLA-A guide RNA comprising a guide sequence selected from (i) SEQ ID NOs: 301-590; or (ii) at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301-428 and 463-511; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 429-462 and 512-590; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301-428 and 463-511; or a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from SEQ ID NOs: 429-462 and 512-590; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Tables 4-7; or (v) a guide sequence complementary to at least 17, 18, 19, or 20 consecutive nucleotides of a genomic region listed in Table 4, Table 5B, or Table 6; or a guide sequence complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a genomic region listed in Table 5A or Table 7; or (vi) a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from (v); and optionally (b) a first RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent; and contacting the cell with (a) a guide sequence selected from (i) SEQ ID NOs: 1-91 and 101-185; or (ii) at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-91; or at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-185; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1-91; or a guide sequence that is at least 95%, 90%, 85%, 80%,A guide sequence that is 75% or 70% identical; or (iv) a guide sequence that binds to a target site comprising a genomic region listed in Table 2 or Table 3; or (v) a guide sequence that is complementary to at least 17, 18, 19, or 20 consecutive nucleotides of the genomic region listed in Table 2, or a guide sequence that is complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of the genomic region listed in Table 3; or (vi) a guide sequence that is at least 95%, 90%, 85%, 80%, 75%, or 70% identical to a sequence selected from (v); and optionally (b) an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent, contacting a cell with a second composition comprising; In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein. In some embodiments, the RNA-guided DNA binding agent is selected from one of S. pyogenes Cas9, Neisseria meningitidis Cas9, such as Nme2Cas9, S. thermophilus Cas9, S. aureus Cas9, Francisella novicida Cpf1, Acidaminococcus sp. Cpf1, Lachnospiraceae bacterium Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, and CasX. In some embodiments, the RNA-guided DNA binding agent is selected from one of S. pyogenes Cas9, Neisseria meningitidis Cas9, such as Nme2Cas9, S. thermophilus Cas9, S. aureus Cas9, Francisella novicida Cpf1, Acidaminococcus sp. Cpf1, Lachnospiraceae bacterium Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, and CasX and comprises a polynucleotide. In some embodiments,The RNA-guided DNA binding agent is S. pyogenes Cas9. In some embodiments, the CIITA guide RNA is an S. pyogenes Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9, such as Nme2Cas9. In some embodiments, the RNA-guided DNA binding agent is S. thermophilus Cas9. In some embodiments, the RNA-guided DNA binding agent is S. aureus Cas9. In some embodiments, the RNA-guided DNA binding agent is Cpf1 from F. novicida. In some embodiments, the RNA-guided DNA binding agent is Cpf1 from Acidaminococcus sp. In some embodiments, the RNA-guided DNA binding agent is Cpf1 from Lachnospiraceae bacterium ND2006. In some embodiments, the RNA-guided DNA binding agent is a C to T base editor. In some embodiments, the RNA-guided DNA binding agent is an A to G base editor. In some embodiments, the base editor comprises a deaminase and an RNA-guided nickase. In some embodiments, the RNA-guided DNA binding agent comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the RNA-guided nickase is SpyCas9 nickase. In some embodiments, the RNA-guided nickase comprises NmeCas9 nickase. In some embodiments, the RNA-guided DNA binding agent is Cas12a. In some embodiments, the RNA-guided DNA binding agent is CasX. In some embodiments, the surface expression of the HLA-A protein (i.e., the engineered cell) is thereby reduced or eliminated.,

[0557] In some embodiments, the method includes a method of gen...

Claims

[Claim 1] The invention described in the specification.

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