Compositions and methods for CD34 genetic modification

Abstract

Provided herein are gRNAs that include a targeting domain that targets CD34, which can be used, for example, to effect modifications in cells. Also provided herein are methods of genetically engineered cells having modifications (e.g., insertions or deletions) in the CD34 gene, and methods that include administering such genetically engineered cells to a subject, such as a subject with a hematopoietic malignancy.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No. 63 / 132,852, filed December 31, 2020, which is incorporated by reference in its entirety herein.

[0002] background When a subject is administered an immunotherapy that targets an antigen associated with a disease or condition, such as an anti-cancer CAR-T therapy, the therapy may deplete not only the pathological cells that are intended to be targeted, but also non-pathological cells that may express the target antigen. This "on-target, off-disease" effect has been reported for some CAR-T therapeutics, such as those that target CD19 or CD33. If the target antigen is expressed on the surface of cells that are required for survival or the subject, or on the surface of cells whose depletion is significantly detrimental to the subject's health, the subject will not be able to receive the immunotherapy or will face severe side effects once such a therapy is administered. Summary of the Invention

[0003] overview Aspects of the present disclosure describe compositions, methods, strategies, and therapeutic modalities that address the deleterious on-target, off-disease effects of certain immunotherapeutic approaches, e.g., immunotherapies involving lymphoid effector cells, e.g., CAR-T cells or CAR-NK cells, that target specific antigens, in a subject in need thereof. The present disclosure is directed, in part, to compositions, methods, strategies, and therapeutic modalities that reduce (e.g., deplete) a population of stem cells (e.g., CD34-expressing hematopoietic stem cells (HSCs)) in a subject using, e.g., immunotherapeutic approaches involving lymphoid effector cells, e.g., CAR-T cells or CAR-NK cells, that target CD34. In some aspects, such compositions, methods, strategies, and therapeutic modalities replace or replenish a population of cells (e.g., hematopoietic cells, hematopoietic stem cells (HSCs)) with engineered cells (e.g., engineered stem cells, e.g., HSCs) that do not express CD34, express reduced levels of CD34 (e.g., compared to wild-type cells), or express a variant form of CD34 that is not recognized by CD34-targeted immunotherapeutic agents. Without wishing to be bound by theory, it is believed that engineered cells (e.g., engineered stem cells, e.g., HSCs) that do not express CD34, express reduced levels of CD34 (e.g., compared to wild-type cells), or express a variant form of CD34 that is not recognized by CD34-targeted immunotherapeutic agents avoid killing by CD34-targeted immunotherapeutic approaches, thus providing a healthy cell population (e.g., stem cell population) to a subject in need thereof to replace a targeted (e.g., malignant) cell population. In some embodiments, immunotherapeutic approaches including, for example, lymphocytic effector cells that target CD34, such as CAR-T cells or CAR-NK cells, and genetically engineered cells (e.g., genetically engineered stem cells, e.g., HSCs) that do not express CD34, express reduced levels of CD34 (e.g., compared to wild-type cells), or express a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34, are administered in combination to a subject in need thereof.

[0004] Thus, aspects of the disclosure provide engineered hematopoietic cells, or progeny thereof, comprising a modified gene encoding CD34. In some embodiments, the modified gene encoding CD34 comprises an indel mutation. In some embodiments, the modified gene encoding CD34 is modified such that an exon is skipped. In some embodiments, exon 1, exon 2, or exon 3 of the modified gene encoding CD34 is engineered. In some embodiments, the modified gene encoding CD34 comprises an insertion or deletion immediately proximal to a site cleaved by an RNA-guided nuclease when bound to a gRNA comprising a targeting domain provided by any of SEQ ID NOs: 11-15. In some embodiments, the modified gene encoding CD34 comprises an insertion or deletion generated by a non-homologous end joining (NHEJ) event or by a homology-directed repair (HDR) event.

[0005] In some embodiments, modification of the gene encoding CD34 alters the expression of CD34 and / or alters the properties of the hematopoietic cells or their progeny. In some embodiments, expression of CD34 is reduced or eliminated compared to a wild-type counterpart cell that does not have a modified gene encoding CD34. In some embodiments, expression of CD34 is less than 25%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% compared to the expression level of CD34 in a wild-type counterpart cell that does not have a modified gene encoding CD34. In some embodiments, the hematopoietic cells, or their progeny, have reduced or no binding to an agent comprising an anti-CD34 binding domain. In some embodiments, the hematopoietic cells, or their progeny, retain the ability to differentiate normally compared to a population of hematopoietic cells that are not genetically engineered. In some embodiments, the modified gene encoding CD34 results in a loss of function of CD34 in the genetically engineered hematopoietic cells, or their progeny.

[0006] In some embodiments, the genetically engineered hematopoietic cell or progeny thereof further comprises an altered gene encoding a lineage-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a lymphocyte-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a myeloid-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a hematopoietic stem cell-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a hematopoietic stem or progenitor cell-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a T cell-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a B cell-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a NK cell-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a basophil-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is an eosinophil-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a neutrophil-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is a monocyte-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is an erythrocyte-specific cell surface antigen. In some embodiments, the lineage-specific cell surface antigen is CD33, CD123, CLL-1, CD19, CD30, CD5, CD6, CD7, CD38, or BCMA. In some embodiments, the lineage-specific cell surface antigen is expressed on the surface of a malignant cell. In some embodiments, the engineered hematopoietic cells, or their progeny, have reduced or no binding to an agent comprising a binding domain that targets the lineage-specific cell surface antigen. In some embodiments, the hematopoietic cells, or their progeny, lack the CD34 epitope or have an altered CD34 epitope.

[0007] Aspects of the present disclosure provide a guide RNA (gRNA) comprising a targeting domain comprising a sequence set forth in Table 1 and Table 2. In some aspects, the gRNA comprises a targeting domain, wherein the targeting domain comprises any one of the sequences of SEQ ID NOs: 1-15. In some embodiments, the gRNA comprises a first complementarity domain, a linking domain, a second complementarity domain that is complementary to the first complementarity domain, and a proximal domain. In some embodiments, the gRNA is a single guide RNA (sgRNA).

[0008] In some embodiments, the gRNA comprises one or more nucleotide residues that are chemically modified. In some embodiments, the gRNA comprises one or more nucleotide residues that comprise a 2'O-methyl moiety. In some embodiments, the gRNA comprises one or more nucleotide residues that comprise a phosphorothioate. In some embodiments, the gRNA comprises one or more nucleotide residues that comprise a ThioPACE moiety.

[0009] Aspects of the present disclosure provide a method of producing a genetically engineered cell, comprising: providing a cell, and contacting the cell with (i) any of the gRNAs described herein; and (ii) an RNA-guided nuclease that binds to the gRNA, such that the gRNA of (i) forms and / or maintains an RNP complex with the RNA-guided nuclease of (ii), forming an RNP complex under suitable conditions for the RNP complex to bind to a target domain in the genome of the cell. In some embodiments, the contacting comprises introducing (i) and (ii) into the cell in the form of a preformed ribonucleoprotein (RNP) complex. In some embodiments, the contacting comprises introducing (i) and / or (ii) into the cell in the form of a nucleic acid encoding the gRNA of (i) and / or the RNA-guided nuclease of (ii). In some embodiments, the nucleic acid encoding the gRNA of (i) and / or the RNA-guided nuclease of (ii) is an RNA, preferably an mRNA or an mRNA analog. In some embodiments, the ribonucleoprotein complex is introduced into the cell via electroporation.

[0010] In some embodiments, the RNA-guided nuclease is a CRISPR / Cas nuclease. In some embodiments, the CRISPR / Cas nuclease is a Cas9 nuclease. In some embodiments, the CRISPR / Cas nuclease is a spCas nuclease. In some embodiments, the Cas nuclease is a saCas nuclease. In some embodiments, the CRISPR / Cas nuclease is a Cpf1 nuclease.

[0011] In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a hematopoietic progenitor cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T lymphocyte.

[0012] Aspects of the present disclosure provide a genetically engineered cell obtained by any of the methods described herein. Aspects of the present disclosure provide a cell population comprising the genetically engineered cells described herein.

[0013] Aspects of the present disclosure provide a cell population comprising engineered cells, wherein the engineered cells comprise a genomic modification consisting of an insertion or deletion immediately proximal to a site cleaved by an RNA-guided nuclease when bound to a gRNA comprising a targeting domain as set forth in any of Tables 1 and 2. In some embodiments, wherein the genomic modification is an insertion or deletion generated by a non-homologous end joining (NHEJ) event. In some embodiments, wherein the genomic modification is an insertion or deletion generated by a homology directed repair (HDR) event. In some embodiments, the genomic modification results in a loss of function of CD34 in engineered cells bearing such genomic modification. In some embodiments, the genomic modification results in a reduction in expression of CD34 to less than 25%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% compared to the expression level of CD34 in a wild type cell of the same cell type not bearing the genomic modification of CD34. In some embodiments, the engineered cells are hematopoietic stem or progenitor cells. In some embodiments, the engineered cells are immune effector cells. In some embodiments, the engineered cells are T lymphocytes. In some embodiments, the immune effector cells express a chimeric antigen receptor (CAR). In some embodiments, the CAR targets CD34.

[0014] Aspects of the disclosure provide methods that include administering to a subject in need thereof any of the genetically engineered cells described herein (e.g., genetically engineered hematopoietic cells or progeny thereof described herein), or any of the cell populations described herein. In some embodiments, the subject has or has been diagnosed with a hematopoietic malignancy. In some embodiments, the method further includes administering to the subject an effective amount of an agent that targets CD34, where the agent comprises an antigen-binding fragment that binds to CD34. In some embodiments, the agent is an antibody-drug conjugate, or an immune effector cell expressing a chimeric antigen receptor (CAR). In some embodiments, the subject has a malignancy associated with or characterized by expression of CD34 on the malignant cell. In some embodiments, the malignant cell is a cancer stem cell. In some embodiments, the subject has a hematopoietic malignancy. In some embodiments, the subject has an autoimmune disease.

[0015] Aspects of the disclosure provide pharmaceutical compositions comprising engineered hematopoietic cells (e.g., engineered HSCs) as described herein. In some embodiments, the pharmaceutical composition further comprises an effective amount of an agent that targets CD34, such as an antigen-binding fragment that binds CD34, an antibody-drug conjugate, or an immune effector cell expressing a chimeric antigen receptor (CAR).

[0016] Aspects of the present disclosure provide engineered hematopoietic stem cells comprising modifications in the gene encoding CD34. In some embodiments, the engineered hematopoietic stem cells do not express native CD34 protein. In some embodiments, the engineered hematopoietic stem cells are functionally indistinguishable from native hematopoietic stem cells expressing CD34. In some embodiments, the engineered hematopoietic stem cells express one or more hematopoietic stem cell markers. In some embodiments, the engineered hematopoietic stem cells express one or more of CD49c, CD71, CD90, CD117, CD135, CD201, CD228, CD243, CD292, CDw293, CD309, CD318, CD325, and CD349. In some embodiments, the engineered hematopoietic stem cells do not express CD34 on their cell surface. In some embodiments, the engineered hematopoietic stem cells do not express a CD34 epitope recognized by an anti-CD34 antibody on their cell surface. In some embodiments, the genetically engineered hematopoietic stem cells do not express CD34 on their cell surface. In some embodiments, the genetically engineered hematopoietic stem cells do not express CD34 on their cell surface, and express one or more of CD71, CD90, CD201, and CD49c. In some embodiments, the genetically engineered hematopoietic stem cells do not express CD34 on their cell surface, and express one or more of CD90, CD201, and CD49c. In some embodiments, the genetically engineered hematopoietic stem cells do not express lineage-specific surface markers characteristic of differentiated hematopoietic cells (lin-). In some embodiments, the genetically engineered hematopoietic stem cells do not express CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, or CD235a, or any combination thereof. In some embodiments, the genetically engineered hematopoietic stem cells are capable of long-term engraftment in a human recipient. In some embodiments, the genetically engineered hematopoietic stem cells are capable of reconstituting the hematopoietic system in a human recipient after engraftment. In some embodiments, the genetically engineered hematopoietic stem cells do not express CD34 on their cell surface, but express CD90, CD201, and CD49c.In some embodiments, the genetically engineered hematopoietic stem cells do not express CD34 on their cell surface, express one or more of CD90, CD201, and CD49c, and are negative for CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD45RA, CD56, CD123, and CD235a. In some embodiments, the genetically engineered hematopoietic stem cells do not express CD34 on their cell surface, express CD90, CD201, and CD49c, and are negative for CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD45RA, CD56, CD123, and CD235a.

[0017] The above summary is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the detailed description, drawings, examples, and claims. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 is a schematic diagram of the CD34 gene showing the coding regions for the two splice variants of human CD34 ("CD34 CDS"). The number of exemplary Cas9 gRNA target sequences within the CD34 gene and their respective binding sites are indicated by short arrows in the lower portion of the panel, and their locations within the named gRNAs CD34-1 through CD34-5 are indicated by short, dark arrows in the center of the panel.

[0019] [Figure 2A] Figures 2A-2C provide schematic diagrams showing the location of exemplary guide RNAs described herein relative to the CD34 gene. Figure 2A is a schematic diagram showing the location of exemplary guide RNAs described herein relative to the nucleic acid sequence encoding exon 1 of CD34. [Figure 2B]FIG. 2B is a schematic diagram showing the location of exemplary guide RNAs described herein relative to the nucleic acid sequence encoding exon 2 of CD34. [Figure 2C] FIG. 2C is a schematic diagram showing the location of exemplary guide RNAs described herein relative to the nucleic acid sequence encoding exon 3 of CD34.

[0020] [Figure 3A] Figures 3A-3D show the viability and editing efficiency of CD34-edited hematopoietic stem cells. Figure 3A is a schematic diagram showing the experimental workflow. CD34+ cells were thawed and electroporated with Cas9 protein and an exemplary CD34-targeting gRNA described herein, or a control gRNA targeting CD5 ("Ctrl"), followed by analysis of cell viability and editing efficiency by sequencing. [Figure 3B] Figure 3B shows the average cell numbers measured 24 and 48 hours after electroporation for the indicated sgRNA conditions. [Figure 3C] FIG. 3C shows cell viability measured 24 and 48 hours after electroporation for the indicated sgRNA conditions. [Figure 3D] FIG. 3D shows the editing efficiency of the CD34 locus as determined by sequencing analysis.

[0021] [Figure 4] Figure 4 shows the indel (insertion / deletion) distribution for HSCs edited with an exemplary CD34-targeting gRNA referred to as guide CD34-2. The X-axis shows the size of the indel and the Y-axis shows the percentage of a particular indel in the mixture.

[0022] [Figure 5A]Figures 5A-5C show the survival and editing efficiency of CD34-edited donor hematopoietic stem cells. Figure 5A is a schematic diagram showing the experimental workflow. CD34+ cells were thawed and electroporated with Cas9 protein and an exemplary CD34-targeting gRNA described herein, followed by analysis of cell viability and editing efficiency by sequencing. [Figure 5B] FIG. 5B shows the average cell numbers measured 24 and 96 hours after electroporation for the indicated gRNA conditions. [Figure 5C] FIG. 5C shows cell viability measured 24 and 96 hours after electroporation for the indicated gRNA conditions.

[0023] [Figure 6A] Figures 6A and 6B show knockdown of CD34 expression in CD34-edited donor hematopoietic stem cells 96 hours after electroporation according to the workflow presented in Figure 5A. Figure 6A presents plots showing CD34 expression in donor hematopoietic stem cells edited with the exemplary CD34-targeted gRNAs shown, mock-edited cells ("Mock"), and cells that were not electroporated ("No EP") by flow cytometry analysis using an anti-CD34-PE antibody. The X-axis shows the intensity of antibody staining (anti-CD34-PE Texas) and the Y-axis corresponds to the number of cells. [Figure 6B] FIG. 6B presents plots showing CD34 expression in donor hematopoietic stem cells edited using the exemplary CD34-targeting gRNAs shown, mock-edited cells ("Mock"), and cells that were not electroporated ("No EP") based on the relative intensity of anti-CD34-PE antibody by flow cytometry analysis.

[0024] [Figure 7A] Figures 7A-7D present flow cytometry analysis plots of CD34-edited donor hematopoietic stem cells 96 hours after electroporation. Figure 7A shows the unedited control population of cells. [Figure 7B]FIG. 7B shows a donor hematopoietic stem cell edited using the exemplary CD34-targeting gRNA guide CD34-2. [Figure 7C] FIG. 7C shows a donor hematopoietic stem cell edited using the exemplary CD34-targeting gRNA guide CD34-3. [Figure 7D] Figure 7D shows donor hematopoietic stem cells edited using the exemplary CD34-targeting gRNA guide CD34-5. The X-axis shows CD34 expression based on binding of an anti-CD34 antibody (anti-CD34 (PE Texas)), and the Y-axis shows live / dead staining (L / D (APC Cy7)).

[0025] [Figure 8] Figure 8 is a schematic diagram showing the workflow of a large-scale editing experiment targeting CD34 in HSCs, measuring the effect of editing 48 hours and 168 hours after editing. CD34+ cells were thawed and electroporated with Cas9 protein and an exemplary CD34-targeting gRNA described herein, followed by collection of cells at 48 hours and 168 hours after electroporation for analysis. Medium was changed at 48 hours and 120 hours.

[0026] [Figure 9A] Figures 9A and 9B show knockdown of CD34 expression in CD34-edited donor hematopoietic stem cells 48 hours after electroporation according to the workflow presented in Figure 8. Figure 9A presents plots showing CD34 expression in donor hematopoietic stem cells edited with the exemplary CD34-targeted gRNAs shown, mock-edited cells ("Mock"), and cells that were not electroporated ("No EP") by flow cytometry analysis using an anti-CD34-PE antibody. The X-axis shows the intensity of antibody staining (anti-CD34-PE Texas) and the Y-axis corresponds to the number of cells. [Figure 9B]FIG. 9B presents plots showing the percentage of CD34+ cells in donor hematopoietic stem cells edited using the exemplary CD34-targeted gRNAs shown, mock-edited cells ("Mock"), and non-electroporated cells ("No EP") based on the relative intensity of anti-CD34-PE antibody by flow cytometry analysis.

[0027] [Figure 10A] Figures 10A and 10B show knockdown of CD34 expression in CD34-edited donor hematopoietic stem cells 168 hours after electroporation according to the workflow presented in Figure 8. Figure 10A presents plots showing CD34 expression in donor hematopoietic stem cells edited with the exemplary CD34-targeted gRNAs shown, mock-edited cells ("Mock"), and cells that were not electroporated ("No EP") by flow cytometry analysis using an anti-CD34-PE antibody. The X-axis shows the intensity of antibody staining (anti-CD34-PE Texas) and the Y-axis corresponds to the number of cells. [Figure 10B] FIG. 10B presents plots showing the percentage of CD34+ cells in donor hematopoietic stem cells edited using the exemplary CD34-targeted gRNAs shown, mock-edited cells ("Mock"), and non-electroporated cells ("No EP") based on the relative intensity of anti-CD34-PE antibody by flow cytometry analysis.

[0028] [Figure 11A] Figures 11A-11Q present charts, graphs, and tables showing colony forming unit (CFU) assays for cell lineage differentiation and colony genotyping, as well as editing frequency assays showing indel size, location, and allele analysis for exemplary guide RNAs. CFU-GEMM: colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte; CFU-GM: colony forming unit-granulocyte, macrophage; CFU-E: colony forming unit-erythroblast; BFU-E: burst forming unit-erythroblast. Figure 11A presents an overview of the experimental approach for CFU assays in CD34-edited HSCs. [Figure 11B] Figures 11B-11C present CFU assay data of CD34-edited HSCs, in which two concentrations of edited cells were seeded and used for CFU analysis after editing using either the CRISPR / Cas system containing either of two exemplary CD34-targeting guide RNAs: CD34-2 (also referred to herein as guide 2, g2, or sgCD34_2) or CD34-3 (also referred to herein as guide 3, g3, or sgCD34_3). [Figure 11C] Figures 11B-11C present CFU assay data of CD34-edited HSCs, in which two concentrations of edited cells were seeded and used for CFU analysis after editing using either the CRISPR / Cas system containing either of two exemplary CD34-targeting guide RNAs: CD34-2 (also referred to herein as guide 2, g2, or sgCD34_2) or CD34-3 (also referred to herein as guide 3, g3, or sgCD34_3). [Figure 11D] Figure 11D and Figure 11E present editing efficiency data in various HSC burst forming units (BFU) and colony forming units (CFU) engineered with CD34-2 and CD34-3, respectively. [Figure 11E] Figure 11D and Figure 11E present editing efficiency data in various HSC burst forming units (BFU) and colony forming units (CFU) engineered with CD34-2 and CD34-3, respectively. [Figure 11F] Figures 11F and 11G present the insertion / deletion (indel) position distribution for CD34-2 and CD34-3 edited HSC colonies, respectively. [Figure 11G] Figures 11F and 11G present the insertion / deletion (indel) position distribution for CD34-2 and CD34-3 edited HSC colonies, respectively. [Figure 11H] Figures 11H-11K show allelic analysis of CD34 editing in HSCs using CD34-2 or CD34-3, respectively. [Figure 11I]Figures 11H-11K show allelic analysis of CD34 editing in HSCs using CD34-2 or CD34-3, respectively. [Figure 11J] Figures 11H-11K show allelic analysis of CD34 editing in HSCs using CD34-2 or CD34-3, respectively. [Figure 11K] Figures 11H-11K show allelic analysis of CD34 editing in HSCs using CD34-2 or CD34-3, respectively. [Figure 11L] Figures 11L and 11M show the distribution of indels in analyzed samples of CD34-2 and CD34-3 edited colonies, respectively, in heatmap format. [Figure 11M] Figures 11L and 11M show the distribution of indels in analyzed samples of CD34-2 and CD34-3 edited colonies, respectively, in heatmap format. [Figure 11N] Figures 11N and 11O show bar graph representations of indel distribution for CD34-edited HSCs edited with CD34-2 and CD34-3, respectively. [Figure 11O] Figures 11N and 11O show bar graph representations of indel distribution for CD34-edited HSCs edited with CD34-2 and CD34-3, respectively. [Figure 11P] Figures 11P and 11Q show the nucleic acid sequence variations, amino acid sequence variations, frequencies, and protein results of the predominant indel species in CD34-2 and CD34-3 edited HSCs, corresponding to the distribution data shown in Figures 11N and 11O, respectively. [Figure 11Q] Figures 11P and 11Q show the nucleic acid sequence variations, amino acid sequence variations, frequencies, and protein results of the predominant indel species in CD34-2 and CD34-3 edited HSCs, corresponding to the distribution data shown in Figures 11N and 11O, respectively.

[0029] [Figure 12A]Figures 12A-12W show graphs of CD34 editing time course data in samples of HSCs from three different donors, and Figures 12A-12C show viability data over 0, 24, 48, 120, 168, and 216 hours post-electroporation for three donor populations of CD34 HSCs (Donor 1, Donor 2, and Donor 3, respectively). [Figure 12B] 12A-12C show viability data for three donor populations of CD34 HSCs (Donor 1, Donor 2, and Donor 3, respectively) over a period of 0, 24, 48, 120, 168, and 216 hours post-electroporation. [Figure 12C] 12A-12C show viability data for three donor populations of CD34 HSCs (Donor 1, Donor 2, and Donor 3, respectively) over a period of 0, 24, 48, 120, 168, and 216 hours post-electroporation. [Figure 12D] Figures 12D-12I show TIDE analysis of editing efficiency in HSCs at various time points after electroporation, expressed as % indels for the predominance of indels compared to control donor HSC samples that were not electroporated or electroporated with control (CTRL) gRNA; Figures 12D-12E show data from editing of Donor 1 HSC, Figures 12F-12G show data from editing of Donor 2 HSC, and Figures 12H-12I show data from editing of Donor 3 HSC. [Figure 12E] Figures 12D-12I show TIDE analysis of editing efficiency in HSCs at various time points after electroporation, expressed as % indels for the predominance of indels compared to control donor HSC samples that were not electroporated or electroporated with control (CTRL) gRNA; Figures 12D-12E show data from editing of Donor 1 HSC, Figures 12F-12G show data from editing of Donor 2 HSC, and Figures 12H-12I show data from editing of Donor 3 HSC. [Figure 12F]Figures 12D-12I show TIDE analysis of editing efficiency in HSCs at various time points after electroporation, expressed as % indels for the predominance of indels compared to control donor HSC samples that were not electroporated or electroporated with control (CTRL) gRNA; Figures 12D-12E show data from editing of Donor 1 HSC, Figures 12F-12G show data from editing of Donor 2 HSC, and Figures 12H-12I show data from editing of Donor 3 HSC. [Figure 12G] Figures 12D-12I show TIDE analysis of editing efficiency in HSCs at various time points after electroporation, expressed as % indels for the predominance of indels compared to control donor HSC samples that were not electroporated or electroporated with control (CTRL) gRNA; Figures 12D-12E show data from editing of Donor 1 HSC, Figures 12F-12G show data from editing of Donor 2 HSC, and Figures 12H-12I show data from editing of Donor 3 HSC. [Figure 12H] Figures 12D-12I show TIDE analysis of editing efficiency in HSCs at various time points after electroporation, expressed as % indels for the predominance of indels compared to control donor HSC samples that were not electroporated or electroporated with control (CTRL) gRNA; Figures 12D-12E show data from editing of Donor 1 HSC, Figures 12F-12G show data from editing of Donor 2 HSC, and Figures 12H-12I show data from editing of Donor 3 HSC. [Figure 12I]Figures 12D-12I show TIDE analysis of editing efficiency in HSCs at various time points after electroporation, expressed as % indels for the predominance of indels compared to control donor HSC samples that were not electroporated or electroporated with control (CTRL) gRNA; Figures 12D-12E show data from editing of Donor 1 HSC, Figures 12F-12G show data from editing of Donor 2 HSC, and Figures 12H-12I show data from editing of Donor 3 HSC. [Figure 12J] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12K] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12L] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12M] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12N]Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12O] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12P] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12Q] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12R] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12S]Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12T] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12U] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12V] Figures 12J-12V show graphs of flow cytometry analysis of CD34 protein surface expression in edited HSCs at the indicated time points; Figures 12J-12N show data from editing of HSCs from donor 1, Figures 12O-12S show data from editing of HSCs from donor 2, and Figures 12T-12V show data from editing of HSCs from donor 3. [Figure 12W] FIG. 12W shows a graph of the average percentage of CD34+ cells over time for two donor HSC samples (Donor 1 and Donor 2) edited using the exemplary guides CD34-2 or CD34-3; error bars represent standard deviation.

[0030] [Figure 13A]13A-13L show graphs assessing myeloid in vitro differentiation (IVD) of CD34-edited human hematopoietic stem cells (HSCs). Figure 13A shows the experimental approach taken to characterize the IVD pattern of CD34-edited HSCs. [Figure 13B] Figure 13B shows gel electrophoresis analysis of PCR amplified genomic DNA isolated 48 hours after electroporation from donor 2 HSCs edited prior to IVD using CD34-2 or CD34-3. [Figure 13C] Figures 13C and 13D show TIDE analysis of % indels in donor 2 HSCs edited with CD34-2 and CD34-3, respectively, 48 hours after electroporation and prior to IVD. [Figure 13D] Figures 13C and 13D show TIDE analysis of % indels in donor 2 HSCs edited with CD34-2 and CD34-3, respectively, 48 hours after electroporation and prior to IVD. [Figure 13E] Figures 13E-13F show flow cytometry analysis of CD34 protein surface expression in edited HSCs 0, 2, or 9 days after electroporation in IVD conditions, as measured in CD34 geometric mean fluorescence intensity of the live population (Figure 13E) and %CD34 positive cells (Figure 13F). [Figure 13F] Figures 13E-13F show flow cytometry analysis of CD34 protein surface expression in edited HSCs 0, 2, or 9 days after electroporation in IVD conditions, as measured in CD34 geometric mean fluorescence intensity of the live population (Figure 13E) and %CD34 positive cells (Figure 13F). [Figure 13G] Figures 13G-13I show flow cytometry analysis of surface expression of cell differentiation markers (monocyte markers CD11b, CD14, granulocyte marker CD15, respectively) in edited HSCs 0, 2, or 9 days after electroporation in IVD conditions, expressed as % surface marker positive cells. [Figure 13H]Figures 13G-13I show flow cytometry analysis of surface expression of cell differentiation markers (monocyte markers CD11b, CD14, granulocyte marker CD15, respectively) in edited HSCs 0, 2, or 9 days after electroporation in IVD conditions, expressed as % surface marker positive cells. [Figure 13I] Figures 13G-13I show flow cytometry analysis of surface expression of cell differentiation markers (monocyte markers CD11b, CD14, granulocyte marker CD15, respectively) in edited HSCs 0, 2, or 9 days after electroporation in IVD conditions, expressed as % surface marker positive cells. [Figure 13J] Figures 13J-13L show flow cytometry analysis of surface expression of cell differentiation markers in edited HSCs 0, 2, or 9 days after electroporation in IVD conditions, expressed as geometric mean fluorescence intensity of surface markers. [Figure 13K] Figures 13J-13L show flow cytometry analysis of surface expression of cell differentiation markers in edited HSCs 0, 2, or 9 days after electroporation in IVD conditions, expressed as geometric mean fluorescence intensity of surface markers. [Figure 13L] Figures 13J-13L show flow cytometry analysis of surface expression of cell differentiation markers in edited HSCs 0, 2, or 9 days after electroporation in IVD conditions, expressed as geometric mean fluorescence intensity of surface markers.

[0031] [Figure 14A] Figures 14A-14B show the in vivo characterization of the CD34-edited mouse model. Figure 14A shows the experimental approach for the in vivo characterization of CD34-edited HSCs in the NGS mouse model. Readouts at different time points include, for example, immunophenotyping of the resulting cells. [Figure 14B] FIG. 14B shows exemplary observations characterizing CD34-edited HSCs in the NGS mouse model following engraftment of edited cells into the blood and bone marrow of mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Some aspects of the present disclosure provide compositions, methods, strategies, and therapeutic modalities relating to genetically modified cells, e.g., hematopoietic cells, that are deficient in the expression of an antigen targeted by a therapeutic agent, e.g., an immunotherapeutic agent. The genetically modified cells provided herein are useful, for example, to reduce or avoid certain undesirable effects associated with certain immunotherapeutic agents, e.g., any on-target, off-disease cytotoxicity.

[0033] Such undesirable effects associated with a particular immunotherapeutic agent may occur, for example, when healthy cells in a subject requiring immunotherapeutic intervention express the antigen targeted by the immunotherapeutic agent. For example, a subject may be diagnosed with a malignancy associated with elevated levels of expression of a particular antigen, which is typically not expressed in healthy cells, but may be expressed at relatively low levels in a subset of non-malignant cells in the subject. Alternatively or additionally, a subject may require ablation of cells expressing a particular antigen, such as CD34. Administration of an immunotherapeutic agent, such as a CAR-T cell therapeutic agent or an antigen-targeting therapeutic antibody or antibody-drug conjugate (ADC) to a subject may result in efficient killing of target cells, but may also result in ablation of non-target cells expressing the antigen in the subject. This on-target, off-disease cytotoxicity may result in significant side effects, but in some cases, completely negates the use of the immunotherapeutic agent.

[0034] The compositions, methods, strategies, and treatment modalities provided herein address the problem of on-target, off-disease cytotoxicity of certain immunotherapeutic agents. The compositions, methods, strategies, and treatment modalities provided herein also provide alternatives to traditional methods of ablating cell populations expressing CD34, such as irradiation. For example, some embodiments of the present disclosure provide engineered cells that have modifications in their genome that result in the lack of expression of the antigen targeted by the immunotherapeutic agent, or a particular form of that antigen. Such engineered cells, and their progeny, are not targeted by the immunotherapeutic agent and thus are not subject to any cytotoxicity caused by the immunotherapeutic agent. Such cells can be administered to a subject receiving an antigen-targeting immunotherapeutic agent, for example, to replace healthy cells that would have been targeted and killed by the cellular therapeutic agent, and / or to provide a population of cells that are resistant to targeting by the cellular therapeutic agent. For example, if healthy hematopoietic cells in a subject express an antigen, the genetically engineered hematopoietic cells provided herein, e.g., genetically engineered hematopoietic stem or progenitor cells, may be administered to a subject that does not express the antigen and thus is not targeted by the cellular therapeutic agent. Such hematopoietic stem or progenitor cells can repopulate the hematopoietic niche in the subject and their progeny can reconstitute various lineages, including those that would have been eliminated by the cellular therapeutic agent.

[0035] CD34 is a 115 kDa transmembrane glycoprotein receptor rich in O- and N-glycans that is typically expressed on the surface of human hematopoietic stem and progenitor cells and endothelial cells. The function of CD34 is not fully characterized, but expression of CD34 is thought to be regulated by growth factors such as TGF-β1 and TNF-α and by oxygen concentration. CD34 has been reported to regulate cell adhesion, cell shape, or be involved in cell migration (e.g., T cell migration). See Tasev et al. Angiogenesis (2016) 19:325-338. In some embodiments, HSCs express CD34. In some embodiments, CD34 is used as a cell surface marker for detection and / or isolation of stem or progenitor cells (e.g., HSCs). - The gene encoding human CD34 contains eight exons and is located on chromosome 1.

[0036] In addition to its expression on HSCs, CD34 expression has also been associated with some hematopoietic malignancies.

[0037] Due to the shared expression of CD34 on both normal and healthy HSCs, as well as being an expressed antigen on malignant cells, therapeutic targeting of CD34 may result in depletion of the healthy stem and / or progenitor cell pool.

[0038] Described herein are gRNAs that have been developed to specifically direct genetic modification of the gene encoding CD34. Also provided herein are uses of such gRNAs to generate genetically modified cells, such as hematopoietic cells, immune cells, lymphocytes, and populations of such cells, that are deficient in CD34 or have reduced expression of CD34 such that the modified cells are not recognized by CD34-specific immunotherapy. Also provided herein are methods that include administering such cells, or compositions thereof, to a subject to address the problem of on-target, off-disease cytotoxicity of certain immunotherapeutic agents. In some examples, as described herein, the genetically modified cells are hematopoietic cells (e.g., HSCs) that are deficient in CD34 or have reduced expression of CD34, and are capable of developing into, for example, progenitor cells or lineage-committed cells. Alternatively or additionally, in some examples, as described herein, the genetically modified cells are immune cells, such as CD34-specific CAR T cells that are deficient in or have reduced expression of CD34 and thus are resistant to fratricidal killing by other CD34-specific CAR T cells.

[0039] definition Antibody: As used herein, the term "antibody" refers to a polypeptide that contains sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, intact antibodies produced in nature are typically tetrameric agents of about 150 kD, containing two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD) that associate with each other into what is commonly referred to as a "Y-shaped" structure. Each heavy chain contains at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the base of the stem of the Y). A short region, known as the "switch", connects the heavy chain variable and constant regions. This "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in an intact antibody. Each light chain contains two domains - an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch". An intact antibody tetramer contains two heavy-light chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to each other, so that the dimers are connected to each other to form a tetramer. Naturally occurring antibodies are also typically glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4).When a native antibody folds, the FR regions form a beta sheet that provides a structural framework for the domain, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space such that they create a single hypervariable antigen-binding site located at the tip of a Y structure. The Fc region of a native antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. The affinity and / or other binding properties of the Fc region for an Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, an antibody produced and / or utilized in accordance with the present invention (e.g., as a component of a CAR) comprises a glycosylated Fc domain, including an Fc domain with modified or engineered glycosylation. In some embodiments, any polypeptide or complex of polypeptides that includes a full immunoglobulin domain sequence as found in a native antibody can be referred to and / or used as an antibody, regardless of whether such polypeptide is produced naturally (e.g., generated by an organism that responds to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody," as used herein, in appropriate embodiments (unless otherwise stated or clear from the context), can refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations.For example, in some embodiments, antibodies utilized in accordance with the present invention are in a format selected from, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, which are used in the broadest sense herein and encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably fragments that exhibit the desired antigen-binding activity). The antibodies described herein may be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds with antibody-like properties, as well as other immunological binding moieties known in the art, including, for example, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIP, single domain antibodies, single chain antibodies, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandabs, DVDs, BiTe, TandAbs, or the like, or any combination thereof. The subunit structures and three-dimensional configurations of different classes of antibodies are known in the art. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if it were produced in nature. In some embodiments, an antibody may include a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.).

[0040] Antigen-binding fragment: "Antigen-binding fragment" refers to a portion of an antibody that binds to the antigen to which the antibody binds. Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Exemplary antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; single domain antibodies; linear antibodies; single chain antibody molecules (e.g., scFv or VHH or VH or VL domains only); and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragments of the antibodies described herein are scFvs. In some embodiments, the antigen-binding fragments of the antibodies described herein are VHH domains only. As with whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies may include at least two different variable domains, in which each variable domain is capable of specifically binding to a separate antigen or to a different epitope of the same antigen.

[0041] Antibody heavy chain: As used herein, the term "antibody heavy chain" refers to the larger of the two polypeptide chains present in all antibody molecules in their native conformations.

[0042] Antibody light chain: As used herein, the term "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules in their native conformations.

[0043] Synthetic antibody: As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0044] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of one or more genes, where these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a gene at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0045] Autologous: As used herein, the term "autologous" refers to any material derived from an individual that is later reintroduced into the same individual.

[0046] Allogeneic: As used herein, the term "allogeneic" refers to any material (eg, a population of cells) derived from different animals of the same species.

[0047] Hyperproliferative disease: As used herein, the term "hyperproliferative disease" refers to a disease characterized by rapid and uncontrolled growth of abnormal cells. Hyperproliferative diseases can be benign or malignant. Malignant diseases are typically characterized by the presence of malignant cells, such as cancer cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. In certain embodiments, the hyperproliferative is a hematopoietic malignancy, such as a myeloid malignancy or a lymphoid malignancy. In some embodiments, the hematopoietic malignancy is acute myeloid leukemia. In some embodiments, the hematopoietic malignancy is a myelodysplastic syndrome.

[0048] Conservative sequence modifications: As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into antibodies compatible with various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody can be replaced with another amino acid residue from the same side chain family, and the altered antibodies can be tested for the ability to bind antigen using the functional assays described herein.

[0049] Costimulatory Ligand: As used herein, the term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, and the like) that specifically binds to a cognate costimulatory molecule on an immune cell (e.g., T lymphocyte), thereby providing a signal that mediates an immune cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), CD28, PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll ligand receptor, and a ligand that specifically binds to B7-H3. Costimulatory ligands also include antibodies that specifically bind to ligands that specifically bind to costimulatory molecules present on immune cells (e.g., T lymphocytes), including, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, among others.

[0050] Cytotoxicity: As used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or damaging a cell. In one embodiment, the cytotoxicity of a metabolically enhanced cell is improved, e.g., the cytolytic activity of an immune cell (e.g., T lymphocyte) is increased.

[0051] Effective amount: As used herein, the "effective amount" described herein refers to a dose that is sufficient to prevent or treat a neoplastic disease, e.g., cancer, in an individual. Amounts effective for therapeutic or prophylactic use will depend, for example, on the stage and severity of the disease or disorder being treated, the age, weight, and general health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the active agent selected, the method of administration, the timing and frequency of administration, the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular active agent, and the desired physiological effect. It will be understood by those skilled in the art that various diseases or disorders may require chronic treatments involving multiple administrations, perhaps using the genetically engineered cells (e.g., CAR cells) of the present disclosure in close temporal proximity to edited hematopoietic stem cells, e.g., as described herein, in each or various rounds of administration.

[0052] For purposes of the present invention, the amount or dose of engineered cells containing a heterologous nucleic acid, including a CAR construct, described herein, should be sufficient to provide a therapeutic or prophylactic response in a subject or animal over a reasonable time frame. For example, the dose should be sufficient to bind an antigen or detect, treat, or prevent cancer for a period of about 2 hours or more from the time of administration, e.g., about 12 to about 24 hours or more. In certain embodiments, the period may be even longer. The dose is determined by the efficacy of the particular engineered cells (e.g., CAR cells) of the present disclosure and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0053] Effector function: As used herein, "effector function" or "effector activity" refers to a specific activity carried out by an immune cell in response to a stimulus of the immune cell. For example, the effector function of a T lymphocyte involves recognizing an antigen and killing a cell expressing the antigen.

[0054] Endogenous: As used herein, "endogenous" refers to any material that is produced from or within a particular organism, cell, tissue, or system.

[0055] Exogenous: As used herein, the term "exogenous" refers to any material that is introduced from or produced outside a particular organism, cell, tissue, or system.

[0056] Expand: As used herein, the term "expand" refers to an increase in number, such as in an increase in the number of cells, e.g., immune cells, e.g., T lymphocytes, B lymphocytes, NK cells, and / or hematopoietic cells. In one embodiment, immune cells, e.g., T lymphocytes, B lymphocytes, NK cells, and / or hematopoietic cells, expanded ex vivo are increased in number compared to the number initially present in the culture. In another embodiment, immune cells, e.g., T lymphocytes, B lymphocytes, NK cells, and / or hematopoietic cells, expanded ex vivo are increased in number compared to other cell types in the culture. In some embodiments, expansion can occur in vivo. The term "ex vivo" as used herein refers to cells that have been removed from a living organism (e.g., a human) and grown outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

[0057] Functional portion: As used herein, the term "functional portion," when used in reference to a CAR, refers to any portion or fragment of a CAR construct of the invention, which portion or fragment retains the biological activity of the CAR construct of which it is a part (the parent CAR construct). Functional portions include, for example, those portions of a CAR construct that retain the ability to recognize a target cell or to detect, treat, or prevent cancer to a similar, equal, or greater extent than the parent CAR construct. In reference to a parent CAR construct, a functional portion can include, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95%, or more of the parent CAR.

[0058] A functional moiety can include additional amino acids at the amino or carboxy terminus of the moiety, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR construct. Desirably, the additional amino acids do not interfere with the biological function of the functional moiety, such as recognizing a target cell, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids provide enhanced biological activity compared to the biological activity of the parent CAR construct.

[0059] Functional variant: As used herein, the term "functional variant" refers to a CAR construct, polypeptide, or protein that has substantial or significant sequence identity or similarity with a parent CAR construct, which functional variant retains the biological activity of the CAR of which it is a variant. Functional variants include, for example, those variants of the CAR constructs described herein (parent CAR constructs) that retain the ability to recognize target cells to a similar, the same, or greater extent than the parent CAR construct. In reference to a parent CAR construct, a functional variant can be, for example, at least about 30%, about 50%, about 75%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent CAR construct. A functional variant may, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise the amino acid sequence of a parent CAR construct with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. A non-conservative amino acid substitution may enhance the biological activity of the functional variant, but the biological activity of the functional variant is increased compared to the parent CAR construct.

[0060] gRNA: The terms "gRNA" and "guide RNA" are used interchangeably throughout and refer to a nucleic acid that facilitates specific targeting or homing of a gRNA / Cas9 molecule complex to a target nucleic acid. A gRNA can be unimolecular (having a single RNA molecule) and is sometimes referred to herein as an sgRNA, or modular (comprising one or more, and typically two separate RNA molecules). A gRNA can bind to a target domain in the genome of a host cell. The gRNA (e.g., its targeting domain) can be partially or fully complementary to the target domain. The gRNA may also include a "scaffold sequence" (e.g., a tracrRNA sequence) that recruits the Cas9 molecule (e.g., by the targeting domain of the gRNA sequence) to a target domain bound to the gRNA sequence. The scaffold sequence may include at least one stem-loop structure and recruits an endonuclease. Exemplary scaffold sequences can be found, for example, in Jinek, et al. I (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Publication WO2014 / 093694, and PCT Publication WO2013 / 176772.

[0061] Heterologous: As used herein, the term "heterologous" refers to a phenomenon occurring in a living system, e.g., a cell, that does not naturally occur in that system. For example, expression of a protein in a cell where the protein does not naturally occur in that cell (e.g., the cell does not naturally encode the protein) can be heterologous expression of the protein. In some embodiments, the heterologous nucleic acid encodes a chimeric antigen receptor construct.

[0062] Immune cell: As used herein, the term "immune cell" is used interchangeably herein with the term "immune effector cell" and refers to a cell involved in an immune response, e.g., promoting an immune reaction. Examples of immune cells include, but are not limited to, T lymphocytes, natural killer (NK) cells, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, or B lymphocytes. A source of immune cells (e.g., T lymphocytes, B lymphocytes, NK cells) can be obtained from a subject.

[0063] Immune response: As used herein, the term "immune response" refers to a cellular and / or systemic response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0064] Nucleic Acid: As used herein, the term "nucleic acid" refers to a polymer of at least three nucleotides. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single stranded and double stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in "peptide nucleic acid". In some embodiments, a nucleic acid comprises one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexon) compared to those in the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns.In some embodiments, nucleic acids can be prepared by isolation from natural sources, enzymatic synthesis (e.g., polymerization based on a complementary template, e.g., in vivo or in vitro, regeneration in recombinant cells or systems, or chemical synthesis). In some embodiments, the nucleic acids are at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length.

[0065] Single chain antibody: As used herein, the term "single chain antibody" refers to an antibody formed by recombinant DNA technology in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids.Various methods for producing single chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0066] Specific binding: As used herein, the term "specific binding" refers to an antigen binding domain, such as an antibody drug or a portion of a chimeric antigen receptor, that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antigen binding domain or antibody drug that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not in itself change the classification of the antigen binding domain or antibody drug as specific. In another example, an antigen binding domain or antibody drug that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the classification of the antigen binding domain or antibody drug as specific. In some examples, the term "specific binding" or "specifically binds" may be used in reference to the interaction of an antigen binding domain or antibody drug, protein, or peptide with a second chemical species, but the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, it means that an antigen binding domain or antibody drug recognizes and binds to a particular protein structure rather than to proteins in general. If an antigen-binding domain or antibody drug is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction involving labeled "A" and an antigen-binding domain or antibody drug may reduce the amount of labeled A bound to the antibody.

[0067] Subject: As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog). In some embodiments, a human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject suffers from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or tumor listed herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit a particular symptom (e.g., clinical symptoms of a disease) or characteristic of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptom or characteristic of the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or treatment is to be administered.

[0068] Target: As used herein, the term "target" refers to a cell, tissue, organ, or site within the body that is the target of a provided method, system, and / or composition, e.g., a cell, tissue, organ, or site within the body that is in need of treatment or that is preferentially bound by a CAR, e.g., as described herein.

[0069] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic effect is achieved by suppression, amelioration, or eradication of a disease state.

[0070] Transfected: As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0071] Transgene: As used herein, the term "transgene" refers to an exogenous nucleic acid sequence contained in the genome of a cell, e.g., a cell in which the nucleic acid sequence does not naturally occur. In some embodiments, a transgene can comprise or consist of a nucleic acid sequence encoding a gene product, e.g., a CAR. In some embodiments, a transgene can comprise or consist of an expression construct, e.g., a nucleic acid sequence encoding a gene product under the control of a regulatory element, e.g., a promoter.

[0072] Treat: As used herein, the terms "treat", "treatment", or "treating" refer to partial or complete reduction, remission, delay in onset, inhibition, prevention, mitigation, and / or reduction in the incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who does not exhibit signs or features of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, treatment may be administered to a subject who exhibits only early or mild signs or features of a disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits established, severe, and / or late signs of a disease, disorder, or condition. In some embodiments, treating may include administering immune cells to the subject, including genetically engineered cells expressing a CAR (e.g., T lymphocytes, B lymphocytes, NK cells), or administering a hematopoietic stem cell graft to the subject, including genetically engineered stem cells.

[0073] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may contain cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a tumor is associated with or is a sign of cancer. In some embodiments, a tumor may be a dispersed tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0074] Genetically engineered cells and related compositions and methods. Some aspects of the present disclosure provide engineered cells that contain modifications in their genome that result in loss of expression of CD34, or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34. In some embodiments, the modification in the genome of the cells is a mutation in the genomic sequence that encodes CD34.

[0075] The term "mutation," as used herein, refers to a change (e.g., an insertion, deletion, inversion, or substitution) in a nucleic acid sequence compared to a reference sequence, e.g., a corresponding sequence of a cell that does not have such a mutation, or a corresponding wild-type nucleic acid sequence. In some embodiments provided herein, a mutation in a gene encoding CD34 results in loss of expression of CD34 in cells that have the mutation. In some embodiments, a mutation in a gene encoding CD34 results in expression of a variant form of CD34 that is not bound by an immunotherapeutic agent that targets CD34, or that is bound at a significantly lower level than the non-mutated CD34 form encoded by the gene. In some embodiments, as provided herein, a cell that has a genomic mutation in the CD34 gene is not bound by, or that is bound at a significantly lower level by, an immunotherapeutic agent that targets CD34, e.g., an anti-CD34 antibody or a chimeric antigen receptor (CAR).

[0076] Some aspects of the present disclosure provide compositions and methods for generating genetically engineered cells as described herein, for example, genetically engineered cells that contain modifications in their genome that result in the loss of expression of CD34 or the expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34. Such compositions and methods provided herein include, but are not limited to, suitable strategies and approaches for genetically engineered cells, for example, by using RNA-guided nucleases, such as CRISPR / Cas nucleases, and suitable RNAs that can bind such RNA-guided nucleases and target them to appropriate target sites in the genome of the cells, resulting in genome modifications that result in the loss of expression of CD34 or the expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34.

[0077] In some embodiments, the genetically engineered cells described herein (e.g., genetically engineered hematopoietic cells, such as, for example, genetically engineered hematopoietic stem or progenitor cells or genetically engineered immune effector cells) are generated via genome editing techniques, which include any technique capable of introducing targeted changes, also referred to as "edits," into the genome of a cell.

[0078] One exemplary suitable genome editing technique is "gene editing," which involves the use of RNA-guided nucleases, e.g., CRISPR / Cas nucleases, to introduce targeted single- or double-stranded DNA breaks in the genome of a cell, thereby triggering cellular repair mechanisms, such as, for example, non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ, also sometimes referred to as "alternative NHEJ" or "alt-NHEJ"), or homology-directed repair (HDR), which typically results in an altered nucleic acid sequence (e.g., via an insertion, deletion, inversion, or substitution of a nucleotide or nucleotide sequence) at or immediately proximal to the site of the nuclease break. Yeh et al. Nat. Cell. Biol. (2019) 21:1468-1478; see, e.g., Hsu et al. Cell (2014) 157:1262-1278; Jasin et al. DNA Repair (2016) 44:6-16; Sfeir et al. Trends Biochem. Sci. (2015) 40:701-714.

[0079] Another exemplary suitable genome editing technique is "base editing," which includes the use of nuclease-impaired or partially nuclease-impaired RNA-guided CRISPR / Cas proteins fused to deaminases that target and deaminate specific nucleobases, e.g., cytosine or adenosine nucleobases of C or A nucleotides, resulting in a C to T nucleotide change, or an A to G nucleotide change, via the cellular mismatch repair machinery. See, e.g., Komor et al. Nature (2016) 533:420-424; Rees et al. Nat. Rev. Genet. (2018) 19(12):770-788; Anzalone et al. Nat. Biotechnol. (2020) 38:824-844;

[0080] Yet another exemplary suitable genome editing technique includes "prime editing," which involves the introduction of new genetic information, e.g., modified nucleotide sequences, into specifically targeted genomic sites using catalytically impaired or partially catalytically impaired RNA-guided nucleases, e.g., CRISPR / Cas nucleases, fused to engineered reverse transcriptase (RT) domains. The Cas / RT fusion can be targeted to a target site in the genome by a guide RNA that also contains a nucleic acid sequence encoding the desired edit and serves as a primer for the RT. See, e.g., Anzalone et al. Nature (2019) 576(7785):149-157.

[0081] The use of genome editing techniques typically features the use of a suitable RNA-guided nuclease, which in some embodiments may be catalytically impaired or partially catalytically impaired, for example, for base editing or prime editing. Examples of suitable RNA-guided nucleases include CRISPR / Cas nucleases. For example, in some embodiments, a suitable RNA-guided nuclease for use in the genetically engineered cell methods provided herein is a Cas9 nuclease, such as spCas9 or saCas9 nuclease. In another example, in some embodiments, a suitable RNA-guided nuclease for use in the genetically engineered cell methods provided herein is a Cas12 nuclease, such as Cas12a nuclease. Exemplary suitable Cas12 nucleases include, but are not limited to, AsCas12a, FnCas12a, other Cas12a orthologs, and Cas12a derivatives, such as the MAD7 system (MAD7™, Inscripta, Inc.), or Alt-R Cas12a (Cpf1) Ultra nuclease (Alt-R® Cas12a Ultra; Integrated DNA Technologies, Inc.). See, e.g., Gill et al. LIPSCOMB 2017. In United States: Inscripta Inc.; Price et al. Biotechnol. Bioeng. (2020) 117(60):1805-1816;

[0082] In some embodiments, the engineered cells described herein (e.g., engineered hematopoietic cells, such as, for example, engineered hematopoietic stem or progenitor cells, or engineered immune effector cells) are generated by targeting an RNA-guided nuclease, such as, for example, a CRISPR / Cas nuclease, such as, for example, a Cas9 nuclease or a Cas12a nuclease, to a suitable target site in the genome of the cell under suitable conditions for the RNA-guided nuclease to bind the target site and cleave the genomic DNA of the cell. A suitable RNA-guided nuclease can be targeted to a specific target site in the genome by a suitable guide RNA (gRNA). Suitable gRNAs for targeting CRISPR / Cas nucleases according to aspects of the present disclosure are provided herein, and exemplary suitable gRNAs are described in more detail elsewhere herein.

[0083] In some embodiments, the CD34 gRNA described herein is complexed with a CRISPR / Cas nuclease, e.g., a Cas9 nuclease. A variety of Cas9 nucleases are suitable for use with the gRNA provided herein to produce genome modifications, e.g., in the CD34 gene, resulting in genome editing according to aspects of the present disclosure. Typically, the Cas nuclease and gRNA are provided in a form and under conditions suitable for the formation of a Cas / gRNA complex that targets a target site on the genome of a cell, e.g., a target site in the CD34 gene. In some embodiments, a Cas nuclease is used that exhibits the desired PAM specificity to target the Cas / gRNA complex to a desired target domain in the CD34 gene. Suitable target domains and corresponding gRNA targeting domain sequences are provided herein.

[0084] In some embodiments, the Cas / gRNA complex is formed, e.g., in vitro, and the target cell is contacted with the Cas / gRNA complex, e.g., via electroporation of the Cas / gRNA complex into the cell. In some embodiments, the cell is contacted separately with a Cas protein and a gRNA, and a Cas / gRNA complex is formed within the cell. In some embodiments, the cell is contacted with a nucleic acid, e.g., DNA or RNA encoding a Cas protein, and / or a nucleic acid encoding a gRNA, or both.

[0085] In some embodiments, the genetically engineered cells provided herein are generated using a suitable genome editing technique, wherein the genome editing technique is characterized by the use of a Cas9 nuclease. In some embodiments, the Cas9 molecule is or is derived from Streptococcus pyogenes (SpCas9), Staphylococcus aureus (SaCas9), or Streptococcus thermophilus (stCas9).Additional suitable Cas9 molecules include Neisseria meningitidis (NmCas9), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces spp., Cyclophilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides spp., Blastopyrrhizobium spp., Brevibacillus laterosporus, Campylobacter coli, and , Campylobacter jejuni (CjCas9), Campylobacter lari, Candidatus puniseispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium acorens, Corynebacterium diphtheriae, Corynebacterium mattorkoti, Dinosauroides shibae, Eubacterium doricum, Gamma proteobacteria, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputum, Helicobacter canadensis, Helicobacter cinaedii, Helicobacter mustela, Iriobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae, Methylocystis spp., Methylocystis trichosporium, Mobiluncus murielis, Neisseria basiliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria spp., Neisseria wadswarti, Nitto These include those of the genus Rosomonas, Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia szygii, Rhodopseudomonas palustris, Rhodoblum spp., Simonsiella muereri, Sphingomonas spp., Sporolactobacillus vinea, Staphylococcus lugdunensis, Streptococcus spp., Subdoligranulum spp., Tistrella mobilis, Treponema spp., or Ferminephorobacter eisenniae, or those derived therefrom.In some embodiments, catalytically impaired or partially impaired variants of such Cas9 nucleases may be used. Additional suitable Cas9 nucleases and nuclease variants will be apparent to those of skill in the art based on this disclosure. The disclosure is not limited in this respect.

[0086] In some embodiments, the Cas nuclease is a naturally occurring Cas molecule. In some embodiments, the Cas nuclease is an engineered, altered, or modified Cas molecule that differs by at least one amino acid residue from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule, or a sequence in Table 50 of PCT Publication WO2015 / 157070, which is incorporated herein by reference in its entirety.

[0087] In some embodiments, a Cas nuclease belonging to class 2 type V of Cas nucleases is used. Class 2 type V Cas nucleases can be further classified as type VA, type VB, type VC, and type VU. See, e.g., Stella et al. Nature Structural & Molecular Biology (2017). In some embodiments, the Cas nuclease is a type VB Cas endonuclease, such as C2c1. See, e.g., Shmakov et al. Mol Cell (2015) 60:385-397. In some embodiments, the Cas nuclease used in the genome editing methods provided herein is a type VA Cas endonuclease, such as Cpf1 (Cas12a) nuclease. See, e.g., Strohkendl et al. Mol. Cell (2018) 71:1-9. In some embodiments, the Cas nuclease used in the genome editing methods provided herein is a Cpf1 nuclease from Prevotella or Francisella, Acidaminococcus (AsCpf1), Lachnospiraceae (LpCpf1), or Eubacterium lectare. In some embodiments, the Cas nuclease is MAD7.

[0088] Both naturally occurring and engineered variants of CRISPR / Cas nucleases are suitable for use according to aspects of the present disclosure, for example, dCas or nickase variants, Cas variants with altered PAM specificity, and Cas variants with improved nuclease activity are encompassed by some embodiments of the present disclosure.

[0089] Some features of some exemplary, non-limiting suitable Cas nucleases are described in more detail herein, without wishing to be bound by any particular theory.

[0090] Native Cas9 nucleases typically comprise two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprises domains as described, for example, in PCT Publication WO2015 / 157070, e.g., in Figures 9A-9B therein (this application is incorporated by reference in its entirety).

[0091] The REC lobe contains an arginine-rich bridge helix (BH), a REC1 domain, and a REC2 domain. The REC lobe appears to be a Cas9-specific functional domain. The BH domain is a long alpha-helix and arginine-rich region, which includes amino acids 60-93 of the sequence of S. pyogenes Cas9. The REC1 domain is involved in the recognition of repeat:anti-repeat duplexes, e.g., gRNA or tracrRNA. The REC1 domain contains two REC1 motifs at amino acids 94-179 and 308-717 of the sequence of S. pyogenes Cas9. These two REC1 domains are separated by the REC2 domain in the linear primary structure, but assemble in the tertiary structure to form the REC1 domain. The REC2 domain, or parts thereof, may also play a role in the recognition of the repeat:anti-repeat duplex. The REC2 domain includes amino acids 180-307 of the sequence of S. pyogenes Cas9.

[0092] The NUC lobe contains a RuvC domain (also referred to herein as a RuvC-like domain), an HNH domain (also referred to herein as a HNH-like domain), and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity with members of the retroviral integrase superfamily and cleaves a single strand, e.g., a non-complementary strand, of a target nucleic acid molecule. The RuvC domain is constructed from three split RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are often commonly referred to in the art as the RuvCI domain, or the N-terminal RuvC domain, RuvCII domain, and RuvCIII domain), at amino acids 1-59, 718-769, and 909-1098, respectively, of the S. pyogenes Cas9 sequence. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, but in the tertiary structure, the three RuvC motifs assemble and form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases and cleaves a single strand, e.g., the complementary strand, of a target nucleic acid molecule. The HNH domain is present between the RuvC II-III motifs and includes amino acids 775-908 of the S. pyogenes Cas9 sequence. The PI domain interacts with the PAM of the target nucleic acid molecule and includes amino acids 1099-1368 of the S. pyogenes Cas9 sequence.

[0093] Crystal structures have been determined for the natural bacterial Cas9 nuclease (see, e.g., Jinek et al., Science, 343(6176):1247997, 2014) and for S. pyogenes Cas9 with guide RNAs (e.g., synthetic fusions of crRNA and tracrRNA) (Nishimasu et al., Cell, 156:935-949, 2014; and Anders et al., Nature, 2014, doi:10.1038 / naturel3579).

[0094] In some embodiments, the Cas9 molecules described herein exhibit nuclease activity that results in the introduction of a double-stranded DNA break in or directly proximal to the target site. In some embodiments, the Cas9 molecule is modified to inactivate one of the catalytic residues of the endonuclease. In some embodiments, the Cas9 molecule is a nickase and produces a single-stranded break. See, e.g., Dabrowska et al. Frontiers in Neuroscience (2018) 12 (75). It has been shown that one or more mutations in the RuvC and HNH catalytic domains of the enzyme can improve Cas9 efficiency. See, e.g., Sarai et al. Currently Pharma. Biotechnol. (2017) 18 (13). In some embodiments, the Cas9 molecule is fused to a second domain, e.g., a domain that modifies DNA or chromatin, e.g., a deaminase or demethylase domain. In some such embodiments, the Cas9 molecule is modified to eliminate its endonuclease activity.

[0095] In some embodiments, the Cas nucleases or Cas / gRNA complexes described herein are administered together with a template for homology directed repair (HDR). In some embodiments, the Cas nucleases or Cas / gRNA complexes described herein are administered without an HDR template.

[0096] In some embodiments, Cas9 nucleases are used that are modified to enhance the specificity of the enzyme (e.g., to reduce off-target effects and maintain robust on-target cleavage). In some embodiments, the Cas9 molecule is an enhanced specificity Cas9 variant (e.g., eSPCas9). See, e.g., Slaymaker et al. Science (2016) 351(6268):84-88. In some embodiments, the Cas9 molecule is a high fidelity Cas9 variant (e.g., SpCas9-HF1). See, e.g., Kleinstiver et al. Nature (2016) 529:490-495.

[0097] A variety of Cas nucleases are known in the art and can be obtained from a variety of sources and / or engineered / modified to modulate one or more activities or specificities of the enzyme. The PAM sequence preferences and specificities of suitable Cas nucleases, such as suitable Cas9 nucleases, such as spCas9 and saCas9, are known in the art. In some embodiments, the Cas nuclease is engineered / modified to recognize one or more PAM sequences. In some embodiments, the Cas nuclease is engineered / modified to recognize one or more PAM sequences that are different from the PAM sequence that the Cas nuclease recognizes without engineering / modification. In some embodiments, the Cas nuclease is engineered / modified to reduce the off-target activity of the enzyme.

[0098] In some embodiments, Cas nucleases are used that are further modified to alter the specificity of endonuclease activity (e.g., to reduce off-target cleavage, decrease endonuclease activity or life span in cells, increase homology-directed recombination, decrease non-homologous end joining). See, e.g., Komor et al. Cell (2017) 168:20-36. In some embodiments, Cas nucleases are used that are modified to alter the PAM recognition or preference of the endonuclease. For example, SpCas9 recognizes the PAM sequence NGG, whereas some variants of SpCas9 that contain one or more modifications (e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9) may recognize variant PAM sequences, e.g., NGA, NGAG, and / or NGCG. As another example, SaCas9 recognizes the PAM sequence NNGRRT, whereas some variants of SaCas9 with one or more modifications (e.g., KKH SaCas9) may recognize the PAM sequence NNRT. In another example, FnCas9 recognizes the PAM sequence NNG, whereas variants of FnCas9 with one or more modifications (e.g., RHA FnCas9) may recognize the PAM sequence YG. In another example, Cas12a nuclease with substitution mutations S542R and K607R recognizes the PAM sequence TYCV. In another example, Cpf1 endonuclease with substitution mutations S542R, K607R, and N552R recognizes the PAM sequence TATV. See, e.g., Gao et al. Nat. Biotechnol. (2017) 35(8):789-792.

[0099] In some embodiments, base editors are used to create genomic modifications that result in loss of expression of CD34 or expression of CD34 variants that are not targeted by immunotherapy. Base editors typically comprise a catalytically inactive or partially inactive Cas nuclease fused to a functional domain, e.g., a deaminase domain. See, e.g., Eid et al. Biochem. J. (2018) 475(11):1955-1964; Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, a catalytically inactive Cas nuclease is referred to as "dead Cas" or "dCas". In some embodiments, the endonuclease comprises an adenine base editor (ABE), e.g., dCas fused to an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises a dCas fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)). In some embodiments, the catalytically inactive Cas molecule has reduced activity, e.g., a nickase.

[0100] Examples of suitable base edits include, but are not limited to, BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Targeted AID, Targeted AID-NG, xBE3, eA3A-BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP. Additional examples of base editing can be found, for example, in U.S. Publication No. 2018 / 0312825A1, U.S. Publication No. 2018 / 0312828A1, and PCT Publication WO2018 / 165629A1, which are incorporated by reference in their entireties.

[0101] Some embodiments of the present disclosure provide guide RNAs that are suitable for targeting, e.g., RNA-guided nucleases provided herein to appropriate target sites in the genome of a cell to result in modifications in the genome of the cell that result in loss of expression of CD34, or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34.

[0102] The terms "guide RNA" and "gRNA" are used interchangeably herein and refer to a nucleic acid, typically an RNA, that is bound by an RNA-guided nuclease and facilitates specific targeting or homing of the RNA-guided nuclease to a target nucleic acid, e.g., a target site in the genome of a cell. A gRNA typically comprises at least two domains: a "binding domain," sometimes also referred to as a "gRNA scaffold" or "gRNA backbone," that mediates binding to an RNA-guided nuclease, and a "targeting domain," that mediates targeting of the gRNA-bound RNA-guided nuclease to a target site. Some gRNAs comprise additional domains, e.g., a complementarity domain, or a stem-loop domain. The structures and sequences of naturally occurring gRNA binding domains and their engineered variants are well known to those of skill in the art. Some suitable gRNAs are unimolecular and comprise a single nucleic acid sequence, while other suitable gRNAs comprise two sequences (e.g., a crRNA and a tracrRNA sequence).

[0103] Some exemplary suitable Cas9 gRNA scaffold sequences are provided herein, and additional suitable gRNA scaffold sequences will be apparent to one of skill in the art based on this disclosure. Such additional suitable scaffold sequences include, but are not limited to, those listed in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Publication WO2014 / 093694, and PCT Publication WO2013 / 176772.

[0104] For example, the binding domain of a naturally occurring spCas9 gRNA typically comprises two RNA molecules, a crRNA (partial) and a tracrRNA. Variants of spCas9 gRNA that comprise only a single RNA molecule that includes both the crRNA and tracrRNA sequences are engineered and covalently linked to each other, for example, via a tetraloop or via a click chemistry type covalent bond, and are generally referred to as "single guide RNA" or "sgRNA". Suitable gRNAs for use with other Cas nucleases, for example with Cas12a nuclease, typically comprise only a single RNA molecule, since the naturally occurring Cas12a guide RNA comprises a single RNA molecule. Suitable gRNAs may thus be unimolecular (having a single RNA molecule) and are sometimes referred to herein as sgRNAs, or modular (comprising one or more, and typically two, separate RNA molecules).

[0105] Suitable gRNAs for targeting target sites in the CD34 gene may contain multiple domains. In some embodiments, for example, in some embodiments where Cas9 nuclease is used, a single molecule sgRNA may contain from 5' to 3' the following: a targeting domain corresponding to a target site sequence in the CD34 gene; a first complementarity domain; Linked domains; a second complementarity domain, which is complementary to the first complementarity domain; proximal domain; and Optionally, the tail domain.

[0106] Each of these domains is now described in more detail.

[0107] The gRNAs provided herein typically comprise a targeting domain that binds to a target site in the genome of a cell. The target site is typically a double-stranded DNA sequence that comprises a PAM sequence and a targeting domain on the same strand as the PAM sequence and directly adjacent to it. The targeting domain of the gRNA typically comprises an RNA sequence that corresponds to the targeting domain sequence, where it resembles the sequence of the targeting domain, occasionally with one or more mismatches, but typically comprises an RNA instead of a DNA sequence. The targeting domain of the gRNA thus base pairs (fully or partially complementary) with the sequence of the double-stranded target site that is complementary to the sequence of the targeting domain, and thus with the strand that is complementary to the strand that contains the PAM sequence. It will be understood that the targeting domain of the gRNA typically does not comprise a PAM sequence. It will be further understood that the location of the PAM can be 5' or 3' of the targeting domain sequence, depending on the nuclease used. For example, the PAM is typically 3' of the target domain sequence for Cas9 nuclease and 5' of the target domain sequence for Cas12a nuclease. For illustrations of the location of the PAM and the mechanism of gRNA binding to a target site, see, e.g., Figure 1 in Vanegas et al., Fungal Biol Biotechnol. 2019;6:6, which is incorporated herein by reference. For additional illustrations and explanations of the mechanism of gRNA targeting RNA-guided nucleases to target sites, see Fu Y et al., Nat Biotechnol 2014 (doi:10.1038 / nbt.2808) and Sternberg SH et al., Nature 2014 (doi:10.1038 / naturel3011), both of which are incorporated herein by reference.

[0108] A targeting domain can include a nucleotide sequence that corresponds to the sequence of a targeting domain, i.e., the DNA sequence immediately adjacent to the PAM sequence (e.g., 5' of the PAM sequence for a Cas9 nuclease, or 3' of the PAM sequence for a Cas12a nuclease). A targeting domain sequence typically includes between 17 and 30 nucleotides and can either correspond perfectly to the targeting domain sequence (i.e., without any mismatched nucleotides) or include one or more, but typically not more than four, mismatches. Because the targeting domain is part of an RNA molecule, a gRNA, it typically includes ribonucleotides, whereas a DNA targeting domain includes deoxyribonucleotides.

[0109] An exemplary illustration of a Cas9 target site comprising a 22 nucleotide targeting domain and a NGG PAM sequence, as well as a gRNA comprising a targeting domain that corresponds perfectly to the targeting domain (and thus base pairs with perfect complementarity to the DNA strand complementary to the strand comprising the targeting domain and PAM) is provided below. From top to bottom and left to right, the sequences correspond to SEQ ID NOs:24-26: [ka]

[0110] An exemplary illustration of a Cas12a target site comprising a 22 nucleotide targeting domain and a TTN PAM sequence, as well as a gRNA comprising a targeting domain that corresponds perfectly to the targeting domain (thus base-pairing with perfect complementarity to the DNA strand complementary to the strand comprising the targeting domain and PAM) is provided below. From top to bottom and left to right, the sequences correspond to SEQ ID NOs: 27, 28, and 26: [ka] In some embodiments, the Cas12a PAM sequence is 5'-TTTV-3'.

[0111] Without wishing to be bound by theory, it is believed that at least in some embodiments, the length and complementarity of the targeting domain with the target sequence contributes to the specificity of the interaction of the gRNA / Cas9 molecular complex with the target nucleic acid. In some embodiments, the targeting domain of the gRNA provided herein is 5-50 nucleotides in length. In some embodiments, the targeting domain is 15-25 nucleotides in length. In some embodiments, the targeting domain is 18-22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In some embodiments, the targeting domain corresponds completely to the targeting domain sequence provided herein, or a portion thereof, without mismatches. In some embodiments, the targeting domain of the gRNA provided herein comprises one mismatch to the targeting domain sequence provided herein. In some embodiments, the targeting domain comprises two mismatches to the targeting domain sequence. In some embodiments, the targeting domain comprises three mismatches to the targeting domain sequence.

[0112] In some embodiments, the targeting domain comprises a core domain and a secondary targeting domain, e.g., as described in PCT Publication WO2015 / 157070, which is incorporated by reference in its entirety. In some embodiments, the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the 3'-most 8 to 13 nucleotides of the targeting domain). In some embodiments, the secondary domain is located 5' to the core domain. In some embodiments, the core domain fully corresponds to the targeting domain sequence, or a portion thereof. In other embodiments, the core domain may comprise one or more nucleotides that are mismatched to corresponding nucleotides of the targeting domain sequence.

[0113] In some embodiments, for example, a Cas9 gRNA is provided, in some embodiments, the gRNA comprises a first complementarity domain and a second complementarity domain, where the first complementarity domain is complementary to the second complementarity domain, and in at least some embodiments has sufficient complementarity to the second complementarity domain to form a double-stranded region under at least some physiological conditions. In some embodiments, the first complementarity domain is 5-30 nucleotides in length. In some embodiments, the first complementarity domain comprises three subdomains, in the 5' to 3' direction: a 5' subdomain, a central subdomain, and a 3' subdomain. In some embodiments, the 5' subdomain is 4-9, e.g., 4, 5, 6, 7, 8, or 9 nucleotides in length. In some embodiments, the central subdomain is 1, 2, or 3, e.g., 1 nucleotide in length. In some embodiments, the 3' subdomain is 3-25, e.g., 4-22, 4-18, or 4-10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. The first complementarity domain can share homology with or be derived from a naturally occurring first complementarity domain. In one embodiment, it has at least 50% homology with a S. pyogenes, S. aureus, or S. thermophilus, first complementarity domain.

[0114] The sequences and arrangements of the above domains are described in more detail in PCT Publication WO2015 / 157070, which is incorporated by reference in its entirety, including pages 88-112 therein.

[0115] The linking domain may serve to link the first complementarity domain with the second complementarity domain of a single gRNA molecule. The linking domain may link the first and second complementarity domains covalently or non-covalently. In some embodiments, the linking is covalent. In some embodiments, the linking domain is or includes a covalent bond inserted between the first complementarity domain and the second complementarity domain. In some embodiments, the linking domain includes one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, nucleotides. In some embodiments, the linking domain includes at least one non-nucleotide bond, e.g., as disclosed in PCT Publication WO2018 / 126176, the entire contents of which are incorporated herein by reference.

[0116] In some embodiments, the second complementarity domain is at least partially complementary to the first complementarity domain, and in one embodiment, has sufficient complementarity with the second complementarity domain under at least some physiological conditions to form a double-stranded region. In some embodiments, the second complementarity domain may include a sequence that lacks complementarity with the first complementarity domain, for example, a sequence that loops out from the double-stranded region. In some embodiments, the second complementarity domain is 5-27 nucleotides in length. In some embodiments, the second complementarity domain is longer than the first complementarity region. In one embodiment, the complementarity domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the second complementarity domain includes three subdomains, which are, in the 5' to 3' direction, a 5' subdomain, a central subdomain, and a 3' subdomain. In some embodiments, the 5' subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In some embodiments, the central subdomain is 1, 2, 3, 4, or 5, e.g., 3 nucleotides long. In some embodiments, the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8, or 9 nucleotides long. In some embodiments, the 5' and 3' subdomains of the first complementarity domain are complementary, e.g., fully complementary, to, e.g., the 3' and 5' subdomains of the second complementarity domain, respectively.

[0117] In some embodiments, the proximal domain is 5-20 nucleotides in length. In some embodiments, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In one embodiment, it has at least 50% homology with a proximal domain from S. pyogenes, S. aureus, or S. thermophilus.

[0118] A wide range of tail domains are suitable for use in gRNAs. In some embodiments, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the tail domain nucleotides are from or share homology with sequences from the 5' end of a naturally occurring tail domain. In some embodiments, the tail domains include sequences that are complementary to each other and form a double-stranded region under at least some physiological conditions. In some embodiments, the tail domain is absent or is 1-50 nucleotides in length. In some embodiments, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In some embodiments, the tail domain has at least 50% homology / identity with a tail domain from S. pyogenes, S. aureus, or S. thermophilus. In some embodiments, the tail domain includes nucleotides at the 3' end that are associated with methods of in vitro or in vivo transcription.

[0119] In some embodiments, the gRNA provided herein comprises: For example, a first strand containing from 5' to 3': a targeting domain, which corresponds to the targeting domain in the CD34 gene; and a first complementarity domain; and For example, a second strand containing from 5' to 3': optionally, a 5' extension domain; a second complementarity domain; proximal domain; and Optionally, the tail domain.

[0120] In some embodiments, any of the gRNAs provided herein comprises one or more chemically modified nucleotides. Chemical modifications of gRNAs have been previously described, and suitable chemical modifications include any modifications that are beneficial for gRNA function and do not measurably increase any undesirable characteristics of a given gRNA, such as off-target effects. Suitable chemical modifications are, for example, those that make the gRNA less susceptible to endo- or exonuclease catalytic activity, including, but not limited to, phosphorothioate backbone modifications, 2'-O-Me-modifications (e.g., at either or both of the 3' and 5' ends), 2'F-modifications, replacement of the ribose sugar with the bicyclic nucleotide-cEt, 3'thioPACE (MSP) modifications, or any combination thereof. Additional suitable gRNA modifications will be apparent to those of skill in the art based on this disclosure, including, but not limited to, those described in, for example, Rahdar et al. PNAS (2015) 112(51) E7110-E7117 and Hendel et al., Nat Biotechnol. (2015); 33(9): 985-989, each of which is incorporated herein by reference in its entirety.

[0121] For example, the gRNAs provided herein may comprise one or more 2'-O modified nucleotides, e.g., 2'-O-methyl nucleotides. In some embodiments, the gRNA comprises 2'-O modified nucleotides, e.g., 2'-O-methyl nucleotides, at the 5' end of the gRNA. In some embodiments, the gRNA comprises 2'-O modified nucleotides, e.g., 2'-O-methyl nucleotides, at the 3' end of the gRNA. In some embodiments, the gRNA comprises 2'-O modified nucleotides, e.g., 2'-O-methyl nucleotides, at both the 5' and 3' ends of the gRNA. In some embodiments, the gRNA is 2'-O-modified, e.g., 2'-O-methyl-modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA. In some embodiments, the gRNA is 2'-O-modified, e.g., 2'-O-methyl-modified at the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O-modified, e.g., 2'-O-methyl-modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O-modified, e.g., 2'-O-methyl-modified at the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA. In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the 3'-terminal nucleotide of the gRNA does not have a chemically modified sugar.In some embodiments, the gRNA is 2'-O-modified, e.g., 2'-O-methyl modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA. In some embodiments, the 2'-O-methyl nucleotide comprises a phosphate linkage to an adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide comprises a phosphorothioate linkage to an adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide comprises a thioPACE linkage to an adjacent nucleotide.

[0122] In some embodiments, the gRNAs provided herein may comprise one or more 2'-O-modified and 3' phosphorus modified nucleotides, such as 2'-O-methyl 3' phosphorothioate nucleotides. In some embodiments, the gRNA comprises a 2'-O-modified and 3' phosphorus modified nucleotide, such as 2'-O-methyl 3' phosphorothioate nucleotides, at the 5' end of the gRNA. In some embodiments, the gRNA comprises a 2'-O-modified and 3' phosphorus modified nucleotide, such as 2'-O-methyl 3' phosphorothioate nucleotides, at the 3' end of the gRNA. In some embodiments, the gRNA comprises a 2'-O-modified and 3' phosphorus modified nucleotide, such as 2'-O-methyl 3' phosphorothioate nucleotides, at the 5' and 3' ends of the gRNA. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' phosphorothioate modified at the 5'-end nucleotide of the gRNA, the second nucleotide from the 5'-end of the gRNA, and the third nucleotide from the 5'-end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' phosphorothioate modified at the 3'-end nucleotide of the gRNA, the second nucleotide from the 3'-end of the gRNA, and the third nucleotide from the 3'-end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' phosphorothioate modified at the 5'-end nucleotide of the gRNA, the second nucleotide from the 5'-end of the gRNA, the third nucleotide from the 5'-end of the gRNA, the 3'-end nucleotide of the gRNA, the second nucleotide from the 3'-end of the gRNA, and the third nucleotide from the 3'-end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' phosphorothioate modified at the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA. In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified.In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' phosphorothioate modified at the 5' end nucleotide of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA.

[0123] In some embodiments, the gRNAs provided herein may comprise one or more 2'-O-modifications and 3' phosphorus modifications, such as 2'-O-methyl 3'thio PACE nucleotides. In some embodiments, the gRNAs comprise 2'-O-modifications and 3' phosphorus modifications, such as 2'-O-methyl 3'thio PACE nucleotides, at the 5' end of the gRNA. In some embodiments, the gRNAs comprise 2'-O-modifications and 3' phosphorus modifications, such as 2'-O-methyl 3'thio PACE nucleotides, at the 3' end of the gRNA. In some embodiments, the gRNAs comprise 2'-O-modifications and 3' phosphorus modifications, such as 2'-O-methyl 3'thio PACE nucleotides, at the 5' and 3' ends of the gRNA. In some embodiments, the gRNAs comprise a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom and one or more non-bridging oxygen atoms are replaced with an acetate group. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3'thio PACE modified at the 5'-end nucleotide of the gRNA, the second nucleotide from the 5'-end of the gRNA, and the third nucleotide from the 5'-end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3'thio PACE modified at the 3'-end nucleotide of the gRNA, the second nucleotide from the 3'-end of the gRNA, and the third nucleotide from the 3'-end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3'thio PACE modified at the 5'-end nucleotide of the gRNA, the second nucleotide from the 5'-end of the gRNA, the third nucleotide from the 5'-end of the gRNA, the 3'-end nucleotide of the gRNA, the second nucleotide from the 3'-end of the gRNA, and the third nucleotide from the 3'-end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' thio PACE modified at the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA. In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified.In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, e.g., 2'-O-methyl 3'thio PACE modified at the 5' end nucleotide of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end, and the fourth nucleotide from the 3' end of the gRNA.

[0124] In some embodiments, the gRNA provided herein comprises a chemically modified backbone. In some embodiments, the gRNA comprises a phosphorothioate linkage. In some embodiments, one or more non-bridging oxygen atoms are replaced with a sulfur atom. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage.

[0125] In some embodiments, the gRNA provided herein comprises a thioPACE linkage. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom and one or more non-bridging oxygen atoms are replaced with an acetate group. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each comprise a thioPACE linkage. In some embodiments, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a thioPACE linkage. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a thioPACE linkage. In some embodiments, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a ThioPACE linkage. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a ThioPACE linkage.

[0126] In some embodiments, the gRNAs described herein comprise one or more 2'-O-methyl-3'-phosphorothioate nucleotides, e.g., at least 1, 2, 3, 4, 5, or 6 2'-O-methyl-3'-phosphorothioate nucleotides. In some embodiments, the gRNAs described herein comprise modified nucleotides (e.g., 2'-O-methyl-3'-phosphorothioate nucleotides) at one or more of the three terminal positions and at the 5' end, and / or at one or more of the three terminal positions and at the 3' end. In some embodiments, the gRNAs may comprise one or more modified nucleotides, e.g., as described in PCT Publication Nos. WO2017 / 214460, WO2016 / 089433, and WO2016 / 164356, which are incorporated by reference in their entireties.

[0127] The CD34-targeting gRNA provided herein can be delivered to cells in any suitable manner. For example, various suitable methods have been described for the delivery of CRISPR / Cas systems, including RNPs containing gRNAs bound to RNA-guided nucleases, and exemplary suitable methods include, but are not limited to, electroporation of RNPs into cells, electroporation of mRNAs and gRNAs encoding Cas nucleases into cells, various protein or nucleic acid transfection methods, and delivery of coding RNA or DNA via viral vectors, such as, for example, retroviral (e.g., lentiviral) vectors. Any suitable delivery method is encompassed by the present disclosure, which is not limited in this respect.

[0128] The present disclosure provides a number of CD34 target sites and corresponding gRNAs that are useful for targeting RNA-guided nucleases to human CD34. Table 1 below illustrates preferred target domains in the human endogenous CD34 gene that can be bound by the gRNAs described herein. The exemplary target sequences of human CD34 shown in Table 1 are, in some embodiments, for use with Cas9 nucleases, such as SpCas9. Table 1. Exemplary Cas9 target site sequences for human CD34 are provided, as well as exemplary gRNA targeting domain sequences useful for targeting such sites. For each target site, the first sequence represents the DNA targeting domain sequence, the second sequence represents its reverse complement, and the third sequence represents an exemplary targeting domain sequence of a gRNA that can be used to target the respective target site. [Table 1]

[0129] The present disclosure provides an exemplary CD34 targeting gRNA, which is useful for targeting RNA-guided nuclease to human CD34.The following Table 2 illustrates the preferred targeting domains for use in gRNA to target Cas9 nuclease to human endogenous CD34 gene.The exemplary target sequence of human CD34 shown in Table 2 is for use with Cas9 nuclease, for example, SpCas9, in some embodiments. Table 2. Exemplary Cas9 targeting domain sequences for gRNAs targeted to human CD34 are provided. [Table 2]

[0130] A representative amino acid sequence of CD34 is provided by UniProtKB / Swiss-Prot accession number P28906-2 shown below. MLVRRGARAG PRMPRGWTAL CLLSLLPSGF MSLDNNGTAT PELPTQGTFS NVSTNVSYQE TTTPSTLGST SLHPVSQHGN EATTNITETT VKFTSTSVIT SVYGNTNSSV QSQTSVISTV FTTPANVSTP ETTLKPSLSP GNVSDLSTTS TSLATSPTKP YTSSSPILSD IKAEIKCSGI REVKLTQGIC LEQNKTSSCA EFKKDRGEGL ARVLCGEEQA DADAGAQVCS LLLAQSEVRP QCLLLVLANR TEISSKLQLM KKHQSDLKKL GILDFTEQDV ASHQSYSQKT LIALVTSGAL LAVLGITGYF LMNRRSWSPT GERLGEDPYY TENGGGQGYS SGPGTSPEAQ GKASVNRGAQ ENGTGQATSR NGHSARQHVV ADTEL (SEQ ID NO: 16)

[0131] A representative DNA sequence for CD34 is provided by NCBI reference sequence NM_001025109.2, shown below. agtgtcttcc actcggtgcg tctctctagg agccgcgcgg gaaggatgct ggtccgcagg ggcgcgcgcg cagggcccag gatgccgcgg ggctggaccg cgctttgctt gctgagtttg ctgccttctg ggttcatgag tcttgacaac aacggtactg ctaccccaga gttacctacc cagggaacat tttcaaatgt ttctacaaat gtatcctacc aagaaactac aacacctagt acccttggaa gtaccagcct gcaccctgtg tctcaacatg gcaatgaggc cacaacaaac atcacagaaa cgacagtcaa attcacatct acctctgtga taacctcagt ttatggaaac acaaactctt ctgtccagtc acagacctct gtaatcagca cagtgttcac caccccagcc aacgtttcaa ctccagagac aaccttgaag cctagcctgt cacctggaaa tgtttcagac ctttcaacca ctagcactag ccttgcaaca tctccacta aaccctatac atcatcttct cctatcctaa gtgacatcaa ggcagaaatc aaatgttcag gcatcagaga agtgaaattg actcagggca tctgcctgga gcaaaataag acctccagct gtgcggagtt taagaaggac agggagagg gcctggcccg agtgctgtgt ggggaggac aggctgatgc tgatgctggg gcccaggtat gctccctgct ccttgcccag tctgaggtga ggcctcagtg tctactgctg gtcttggcca agaacaaga aatttccagc aaactccaac ttatgaaaaa gcaccaatct gacctgaaaa agctggggat cctagatttc actgagcaag atgttgcaag ccaccagagc tattcccaaa agaccctgat tgcactggtc acctcgggag ccctgctggc tgtcttgggc atcactggct atttcctgat gaatcgccgc agctggagcc ccacaggaga aaggctggggc gaaccctt attacacgga aaacggtgga ggccagggct atagctcagg acctgggacc tcccctgagg ctcagggaaa ggccagtgtg aaccgagggg ctcaggaaaa cgggaccggc caggccacct ccagaaacgg ccattcagca agaacacg tggtggctga taccgaattg tgactcggct aggtggggca aggctgggca gtgtccgaga gagcacccct ctctgcatct gaccacgtgc tacccccatg ctggaggtga catctcttac gcccaaccct tccccactgc acacacctca gaggctgttc ttggggccct acaccttgag gaggggcagg taaactcctg tcctttacac attcggctcc ctggagccag actctggtct tctttgggta aacgtgtgac gggggaaagc caaggtctgg agaagctccc aggaacaatc gatggccttg cagcactcac acaggacccc cttcccctac cccctcctct ctgccgcaat acaggaaccc ccaggggaaa gatgagcttt tctaggctac aattttctcc caggaagctt tgatttttac cgtttcttcc ctgtattttc tttctctact ttgaggaaac caaagtaacc ttttgcacct gctctcttgt aatgatatag ccagaaaaac gtgttgcctt gaaccacttc cctcatctct cctccaagac actgtggact tggtcaccag ctcctccctt gttctctaag ttccactgag ctccatgtgc cccctctacc atttgcagag tcctgcacag ttttctggct ggagcctaga acaggcctcc caagttttag gacaaacagc tcagttctag tctctctggg gccacacaga aactcttttt gggctccttt ttctccctct ggatcaaagt aggcaggacc atgggaccag gtcttggagc tgagcctc acctgtactc ttccgaaaaa tcctcttcct ctgaggctgg atcctagcct tatcctctga tctccatggc ttcctcctcc ctcctgccga ctcctgggtt gagctgttgc ctcagtcccc caacagatgc ttttctgtct ctgcctccct caccctgagc cccttccttg ctctgcaccc ccatatggtc atagcccaga tcagctccta acccttatca ccagctgcct cttctgtggg tgacccaggt ccttgtttgc tgttgatttc tttccagagg ggttgagcag ggatcctggt ttcaatgacg gttggaaata gaaatttcca gagaagagag tattgggtag atattttttc tgaatacaaa gtgatgtgtt taatactgc aattaaagtg atactgaaac acatctgtta tgtgactctg tcttagctgg gtgtgtctgc atgcaagagt gacaccctcc attagaccta gctagactgt gcagtgatgt ggtggggagg accagccagg gaagagggag cacctcagca gacacaggca ccagccagga tgctaaggac ctttagccaa gtctgccaac tattctcctc catggggaga ggaaacatcc atttccagtg gtagaaaggc agacccgaat gtaccaggga gcttccaaat ggagggtggt atgttgggtt cttaggagct gtacccttca tgaacaccct tctgagaaga ggagcatgct gatcactgct gcaaaatatg caaaacaaag ggaaggggca atgtcctgtg caccctttat tatcaggcca cccccctccc cagcccccca ggtcagagta gacacagtga aggactatgt ggggactgtt gttctagaga cctggcagcc aactcaggga gggggctggt ttccaccctc aactcagccta attaaaaaa aaatctgtaa gcatgtacct ccccccagct tccaaaacaa cccccacccc acccctacca ggccatagga agttggggag ggagtgctga ggagctccag gaaacactcc caagtgtgtc gacagtggca gaggcagttg gggccaaaca aaggttgatt cttccattct tatctccata aagccagacc tttcccttca gcactcctcc acccccatct ccttcttgct tttctccaac tcctctaatc ataggttctt ccctaggaca gaggggaggc gaaatgatga ggttcagagt cttccctcaa aggcgatggc tgccttgagg gttggagcaa aggcgatgcgcaaaagacg atggtaatca gtagggaagt ccagcccact tgcatctagt tgcacatctt gccttgagag taatccagtg agggtctgtc ccagctagga catcaagtag gaggggtggg ttcagggttc agattcctag gaaatatggg aggagagga aaggcaactt ggatgcacct ccagcttcag gcctagcaac ctgcaatgca tccaccctg agtttgctg aatgtgtag tatgctttgg gaggaagggc tgtgtgtgta tgcgggtg gggtggggca gctggttccc tctgacagct ggacagcttg ccctgaagaa ttgctgct ttctggaaa atccacttt cccaccgtgg gcctgagcgt cctggtacag caatggcgcc acctgctggc cttattgagg tcctactgct cagcctcagc tcaatcgcct ccatgttggg cttctctccc tggctgcccc acccttagt ccaatttctc ttgtacaca agctcatata actatagaac gtcactgttg agagaactt taaagataca ttataaa ctcccttatg gtatagttaa agacaacta aggctcagag aagggaggtg gcttgcccaa tcaccagaa ttccaagtc ctgaatctgt agttttccct tccatcatat catcctactc ttctgccgag tcctccgtgt tactccagtt ggatgtcatg aagccagtgt ggcagtgtga agataggttt gggacttcac tctggagca ttcatcac ataagctatc ctaggcctgg ccagccaagc aggtcctgga ggagccccag ccaagatc acaggaggcc atgaggttcg gctcttcgg cgcccacagt gagcccagga aattagctg taggtatta cactgttgac tatggagc atatctggaa tattcttcag ccagattttc atctgaatgg ataaatggga ataccatcta agtccagata aatagatcac ttccatctca tcccttctag gtagattaat cccacacttc ctcttcacac aaaaccagta ataggtcatc gattttgtgc aacaggatgc tgcttctct cctaaagccc ccatcgaaga ggcttccagc caccattcaa tcattcatca agtcttatga tgtgccagac actgcgcgaa atgtgccaga acatctgtta tgtgccagac actgttcttg agactgggga tacagcaaac actcatgaag cttataattc tagcagaaga ggacagtaaa caatgtcatc tcagtaagta tatacatgtg ttttcaggat tgagagctat gaaaaacata aatatattg agaataatgg ttggtatttt acatatggtg gttactttta gaaaaataac agtggagagc acagcttcac ttgaatgaag tggagaagca ggttgtatgc caagctggga gagattatcc cacacagggg aaaggacaag tgcaaagccc tatgatgaaa agctgccaag tgcagaaagc ctcagatggc agggggcaag atggccatga ggttgtgtca gtgagtgggg gtggggagag gcaggaggtc agactacatg gggcctttt agttgtagat tgggaagcca ctggagggtt ttgagcagag aagtcatatc atctgcttta tgttttaaa ggatcatgct ggctggag tagatag aggttgaggg atagaaagt agaagagac cgtagcaga agaacgatca tggctgggag caggtgatca tattggcagt gatgagatca agcagaatttc aaaaagtggt ttcaagtag aggtaacagg acttgctcag tctatttatt tctcaata atatcaat ttacaatgat agtagctaac agttttgag tgcttactgt atgaaaattg agatatggtg ccaatattta atagcatat ttacttaac ttacacaga accctgtgaa gtaggttcta ttactcaga aaagaaact gaaactcaga gaatacaag ggactgtgtt acgtgcacag tggcagaggc aagatgaat aggatgtgag tttattgaa cccaatgt ttaaatctg gggataatac aacacacatt taaaaaaaaaaaaaaaaatgcaca ahaaaagtg agaaaaca cgagaaga ctaaatgaag tgcttgtat ctagatgtgg gcaggaccct ttccagctga gaagatctga gactgggtca tgaacaggtg gtttctgagt gggtcctgta aaatgaata cgattttgat gatagtaatg agtaggaca tttgagactg atagaagt acatacaata tgtagtgatg gggaaagata aggtactgtc aaggacaat gtgttttctg gtagacaga gaagtagaat gtgttaaggg aagccgagta ccagaagat ccggtgtca cagttgtgt agggtgttta aagctaaacc acgagttta atttttacca atagaaggg agccacagaa gagttttccat ttattcatta atttattcat ttattcaaa atatttgag tgctttat aagccaggta ctatgccagg cacctgggat agacatagt cccttctgtc aagtctttac attgggtgga tgtgggaggg acatgaca gaacaatg cattgagtgt aagtgctatg gtataggag ctctgagtgg gaggggcatg gaagccgtgg agaccatgg aaggctccc aggaagtg acgtctggac tgatcctttg gtcaagcagg agttaagag gagaaaagga gagatatggg tgttcccgag agaggaaa gccttgtccc aggagcaag tgaggtgat tgttccagaa atgtgagtga ttctttaag gctcaagcaa agcatgtgat tctctttat accttctatt tctttgctga gtgttctgt tctttgttt caagcatgct gcaattgctc attaaagcat gtttatgatg gctgtctgtt ttaaaattct tgtcagatgg ttcacatc tttacatct caatgttggc atctgttaat gttttttct caatcaatt gagattttcc tggttctttgg tattaccagt gattttaatt gcatctgga atttgggatt tatgttgaaa gactggatct tattgaaaga ttctgtttag cacccctcct ttgataccac actggtgggt ccaggttccc cattcagctg ttgacacctt cagggcagag aggtgggatg gggtgaaggg ggtacctcat tattgctggc ccaggttaga agttcaggct tcccagtaga tctctgctga taccaccctg gtgccatgtc attccttgag tccaaaagtc cctcccaatt ctgccttctt ctctctacat atcggagtct ccctatgttt gacttatata taatgtccag ggttttga gttagttaac aggaggcata agaaaaagtg tgtccactcc atcttgtctg gaactggaag ttcaagtcga atataagaga rich aattacaagc catgagactg rich caggttctac accagctatt ctcaaagccc tcttacactc ttaaaaattt agaacttcaa agagcttttg attttgaaag ttacatctat caattattac tgtttcaaaa attaaattg agaaaatttt atttattaat ttgtttaaaa father ttattcaatt acatgataat gtaagtaatg cttttcttaa tgaaaaata ttatattttc caaacaaaa acaattagga aaaagagtgt cattgtttta gactttggta aatctctcta atatctggct gaagagaaga atgctgattc tttttttt tttttttt tgagacggag tctcgctctg tcacccaggc tggagtgtag tggtgtgatc tcggctcact gcaagctctg cctcccgggt tcacgccatt ctcctgcctc agcctcccaa gtagctggga ctacaggcac ccgccaccac gcccggctaa ttttttgta tttttagtag agatggggtt tcaccgtgtt agccaggctg gtctcgatct cctgacctca tgatccaccc acctcagcct cccaaagcgc tgggattaca ggtgtgagac accgcgccca gccccgaat gctgattct ttatctgctt ctgtattcaa tctgttgtga tatgatgggt agcctctgaa acactccact gtatacttgt gaaagaatga atgtgaaaaa ggaaataga tttgtagtat tattattcaa attgttttga cctcagagac cacttggaaa tgttttaggg aaccccaga ggaccttgga tcatgctttg agaaccgcgg ctctagatat gttactattt cagtagcatc taagtacatg tggctgctga gcacttgtaa tgtggctagt gcaaatgaga gacaggactt ccagctatat gtaatttaat aaactcaaat ttaaaaactg gaacctcata aaatgttttg ttgttgttgt taaacatgac cttatagttt tggtaggaa (SEQ ID NO:17)

[0132] A further representative DNA sequence of CD34 is provided by the NCBI reference sequence number NM_001773.3 shown below. agtgtcttcc actcggtgcg tctctctagg agccgcgcgg gaaggatgct ggtccgcagg ggcgcgcgcg cagggcccag gatgccgcgg ggctggaccg cgctttgctt gctgagtttg ctgccttctg ggttcatgag tcttgacaac aacggtactg ctaccccaga gttacctacc cagggaacat tttcaaatgt ttctacaaat gtatcctacc aagaaactac aacacctagt acccttggaa gtaccagcct gcaccctgtg tctcaacatg gcaatgaggc cacaacaaac atcacagaaa cgacagtcaa attcacatct acctctgtga taacctcagt ttatggaaac acaaactctt ctgtccagtc acagacctct gtaatcagca cagtgttcac caccccagcc aacgtttcaa ctccagagac aaccttgaag cctagcctgt cacctggaaa tgtttcagac ctttcaacca ctagcactag ccttgcaaca tctcccacta aaccctatac atcatcttct cctatcctaa gtgacatcaa ggcagaaatc aaatgttcag gcatcagaga agtgaaattg actcagggca tctgcctgga gcaaaataag acctccagct gtgcggagtt taagaaggac aggggagagg gcctggcccg agtgctgtgt ggggaggagc aggctgatgc tgatgctggg gcccaggtat gctccctgct ccttgcccag tctgaggtga ggcctcagtg tctactgctg gtcttggcca acagaacaga aatttccagc aaactccaac ttatgaaaaa gcaccaatct gacctgaaaa agctggggat cctagatttc actgagcaag atgttgcaag ccaccagagc tattcccaaa agaccctgat tgcactggtc acctcgggag ccctgctggc tgtcttgggc atcactggct atttcctgat gaatcgccgc agctggagcc ccacaggaga aaggctggag ctggaaccct gaccactctt caggaagaaa ggagtctgca catgcagctg caccctccct ccgatccttc ctcccacctc cccctccccc ttctcccacc cctgccccca cttcctgttt gggcccctct cccatccagt gtctcacagc cctgcttacc agataatgct actttattta tacactgtct agggcgaaga cccttattac acggaaaacg gtggaggcca gggctatagc tcaggacctg ggacctcccc tgaggctcag ggaaaggcca gtgtgaaccg aggggctcag gaaaacggga ccggccaggc cacctccaga aacggccatt cagcaagaca acacgtggtg gctgataccg aattgtgact cggctaggtg gggcaaggct gggcagtgtc cgagagagca cccctctctg catctgacca cgtgctaccc ccatgctgga ggtgacatct cttacgccca acccttcccc actgcacaca cctcagaggc tgttcttggg gccctacacc ttgaggaggg gcaggtaaac tcctgtcctt tacacattcg gctccctgga gccagactct ggtcttcttt gggtaaacgt gtgacggggg aaagccaagg tctggagaag ctcccaggaa caatcgatgg ccttgcagca ctcacacagg acccccttcc cctaccccct cctctctgcc gcaatacagg aacccccagg ggaaagatga gcttttctag gctacaattt tctcccagga agctttgatt tttaccgttt cttccctgta ttttctttct ctactttgag gaaaccaaag taaccttttg cacctgctct cttgtaatga tatagccaga aaaacgtgtt gccttgaacc acttccctca tctctcctcc aagacactgt ggacttggtc accagctcct cccttgttct ctaagttcca ctgagctcca tgtgccccct ctaccatttg cagagtcctg cacagttttc tggctggagc ctagaacagg cctcccaagt tttaggacaa acagctcagt tctagtctct ctggggccac acagaaactc tttttgggct cctttttctc cctctggatc aaagtaggca ggaccatggg accaggtctt ggagctgagc ctctcacctg tactcttccg aaaaatcctc ttcctctgag gctggatcct agccttatcc tctgatctcc atggcttcct cctccctcct gccgactcct gggttgagct gttgcctcag tcccccaaca gatgcttttc tgtctctgcc tccctcaccc tgagcccctt ccttgctctg cacccccata tggtcatagc ccagatcagc tcctaaccct tatcaccagc tgcctcttct gtgggtgacc caggtccttg tttgctgttg atttctttcc agaggggttg agcagggatc ctggtttcaa tgacggttgg aaatagaaat ttccagagaa gagagtattg ggtagatatt ttttctgaat acaaagtgat gtgtttaaat actgcaatta aagtgatact gaaacaca (SEQ ID NO: 18)

[0133] Some aspects of the present disclosure provide engineered cells that contain modifications in their genome that result in loss of expression of CD34 or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34. In some embodiments, the modification in the genome of the cells is a mutation in the genomic sequence that encodes CD34. In some embodiments, the modification is effected via genome editing, for example, using a Cas nuclease and a gRNA that targets a CD34 target site provided herein or includes a targeting domain sequence provided herein.

[0134] While the compositions, methods, strategies, and therapeutic modalities provided herein may be applied to any cell or cell type, some exemplary cells and cell types that are particularly suitable for genomic modification in the CD34 gene according to aspects of the invention are described in more detail herein. Those of skill in the art will recognize, however, that the provision of such examples is for purposes of illustrating some specific embodiments, and that additional suitable cells and cell types will be apparent to those of skill in the art based on the present disclosure, which is not limited in this respect.

[0135] Some aspects of the present disclosure provide genetically engineered hematopoietic cells that contain modifications in their genome that result in loss of expression of CD34 or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34. In some embodiments, the genetically engineered cells that contain modifications in their genome result in reduced cell surface expression of CD34 and / or reduced binding by immunotherapeutic agents that target CD34, for example, compared to hematopoietic cells of the same cell type but that do not contain the genomic modification (e.g., HSCs). In some embodiments, the hematopoietic cells are hematopoietic stem cells (HSCs). In some embodiments, the hematopoietic cells are hematopoietic progenitor cells (HPCs). In some embodiments, the hematopoietic cells are hematopoietic stem or progenitor cells. As used herein, HSCs refer to cells that are capable of self-renewal and can generate and / or reconstitute all lineages of the hematopoietic system. In some embodiments, the HSCs can be engrafted into a subject, where they can proliferate and generate and / or reconstitute all lineages of the hematopoietic system. In some embodiments, the HSCs express one or more cell surface markers, e.g., CD34. In some embodiments, the engineered cells described herein (e.g., engineered HSCs) do not express one or more cell surface markers typically associated with the identification or isolation of HSCs, express reduced amounts of the cell surface marker, or express variant cell surface markers that are not recognized by immunotherapeutic agents that target the cell surface markers, but are nevertheless capable of self-renewal and generating and / or reconstituting all lineages of the hematopoietic system.

[0136] In some embodiments, hematopoietic cells (e.g., HSCs or HPCs) that contain modifications in their genome that result in loss of expression of CD34 or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents targeting CD34 are generated using nucleases and / or gRNAs targeting human CD34 as described herein. It will be understood that such cells can be generated by contacting cells with nucleases and / or gRNAs, or the cells can be daughter cells of cells contacted with nucleases and / or gRNAs. In some embodiments, the cells described herein (e.g., genetically engineered HSCs or HPCs) are capable of populating the HSC or HPC niche and / or reconstituting the hematopoietic system of a subject. In some embodiments, the cells described herein (e.g., HSCs or HPCs) are capable of one or more (e.g., all) of engrafting, producing myeloid lineage cells, and producing lymphoid lineage cells in a human subject. In some preferred embodiments, the genetically engineered hematopoietic cells provided herein, or their progeny, are preferably capable of differentiating into all blood cell lineages without any differentiation bias compared to hematopoietic cells of the same cell type, but do not contain genomic modifications that result in loss of expression of CD34, or expression of variant forms of CD34 that are not recognized by immunotherapeutic agents that target CD34.

[0137] In some embodiments, the genetically engineered cells provided herein contain only one genomic modification, such as a genomic modification that results in the loss of expression of CD34, or the expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34. It will be understood that the gene editing methods provided herein may result in a genomic modification in one or both alleles of a target gene. In some embodiments, genetically engineered cells that contain genomic modifications in both alleles of a given locus are preferred.

[0138] In some embodiments, the genetically engineered cells provided herein contain more than one genomic modification, e.g., one or more genomic modifications in addition to a genomic modification that results in loss of expression of CD34, or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34.

[0139] In some embodiments, the genetically engineered cells provided herein comprise a genomic modification that results in loss of expression of CD34 or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34, and further comprise an expression construct encoding a chimeric antigen receptor, e.g., in the form of an expression construct encoding a CAR integrated into the genome of the cell. In some embodiments, the CAR comprises a binding domain, e.g., an antibody fragment, that binds to CD34.

[0140] Some aspects of the present disclosure provide engineered immune effector cells that contain modifications in their genome that result in loss of expression of CD34 or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34. In some embodiments, the immune effector cells are lymphocytes. In some embodiments, the immune effector cells are T lymphocytes. In some embodiments, the T lymphocytes are alpha / beta T lymphocytes. In some embodiments, the T lymphocytes are gamma / delta T lymphocytes. In some embodiments, the immune effector cells are natural killer T (NKT) cells. In some embodiments, the immune effector cells are natural killer (NK) cells. In some embodiments, the immune effector cells do not express an endogenous transgene, e.g., a transgenic protein. In some embodiments, the immune effector cells express a chimeric antigen receptor (CAR). In some embodiments, the immune effector cells express a CAR that targets CD34. In some embodiments, the immune effector cells do not express a CAR that targets CD34.

[0141] In some embodiments, the genetically engineered cells provided herein contain genomic modifications that result in loss of expression of CD34 or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34, and do not contain an expression construct encoding an exogenous protein, e.g., do not contain an expression construct encoding a CAR.

[0142] In some embodiments, the engineered cells provided herein do not substantially express CD34 protein, e.g., do not express CD34 protein that can be measured by a suitable method, such as, for example, an immunostaining method. In some embodiments, the engineered cells provided herein do not substantially express wild-type CD34 protein, but do express a mutant CD34 protein variant, e.g., a variant that is not recognized by an immunotherapeutic agent that targets CD34, e.g., a CAR-T cell therapeutic, or an anti-CD34 antibody, antibody fragment, or antibody-drug conjugate (ADC).

[0143] In some embodiments, the genetically engineered cells provided herein are hematopoietic cells, e.g., hematopoietic stem cells. Hematopoietic cells are typically characterized by pluripotency, self-renewal properties, and / or the ability to generate cells of the hematopoietic lineage. In some embodiments, hematopoietic stem cells (HSCs) can give rise to both myeloid and lymphoid progenitor cells, which further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. In some embodiments, HSCs are characterized by expression of the cell surface marker CD34 (e.g., CD34+). In some embodiments, CD34 can be used to identify and / or isolate HSCs. In some embodiments, the absence of one or more cell surface markers, e.g., CD34, is associated with lineage commitment. In some embodiments, the engineered cells described herein (e.g., engineered HSCs) do not express CD34, express CD34 at reduced levels, or express a CD34 variant (e.g., that is not recognized by immunotherapeutic agents that target CD34). In some such embodiments, for example, in embodiments where the engineered HSCs are characterized by a complete loss of expression of CD34, the engineered cells are not identifiable or isolatable as HSCs by expression of CD34. In some embodiments, the engineered HSCs do not express CD34, express CD34 at reduced levels, or express a CD34 variant (e.g., that is not recognized by immunotherapeutic agents that target CD34), but are nevertheless HSCs capable of self-renewal.

[0144] In some embodiments, engineered HSCs disclosed herein, e.g., HSCs that do not express CD34, express CD34 at reduced levels, or express a CD34 variant, are identifiable or isolatable as HSCs by expression of CD34, but can still be identified as HSCs, e.g., by other characteristics of HSCs. For example, in some embodiments, engineered HSCs disclosed herein, e.g., HSCs that do not express CD34, express CD34 at reduced levels, or express a CD34 variant, can be identified as HSCs by the status of their CD34 promoter activity or the epigenetic status of the CD34 promoter in their genome, e.g., via a heterologous reporter construct driven by the endogenous CD34 promoter, or via the methylation status of the CD34 promoter (or other suitable epigenetic promoter). In some embodiments, a genetically engineered HSC disclosed herein, e.g., HSC that does not express CD34, expresses reduced levels of CD34, or expresses a CD34 variant, can be identified as an HSC by its expression profile of other HSC markers, e.g., in that such expression profile matches or closely resembles that of a non-edited HSC. In some embodiments, a genetically engineered HSC disclosed herein, e.g., HSC that does not express CD34, expresses reduced levels of CD34, or expresses a CD34 variant, can be identified as an HSC by its ability to engraft and reconstitute all hematopoietic cell lineages in a recipient subject, e.g., a human subject in need thereof.

[0145] In some embodiments, the population of genetically engineered cells described herein comprises a plurality of genetically engineered hematopoietic stem cells. In some embodiments, the population of genetically engineered cells described herein comprises a plurality of genetically engineered hematopoietic progenitor cells. In some embodiments, the population of genetically engineered cells described herein comprises a plurality of genetically engineered hematopoietic stem cells and a plurality of genetically engineered hematopoietic progenitor cells.

[0146] In some embodiments, the genetically engineered HSCs are obtained from a subject, such as a human subject. Methods for obtaining HSCs are described, for example, in PCT Application No. US2016 / 057339, which is incorporated herein by reference in its entirety. In some embodiments, the HSCs are peripheral blood HSCs. In some embodiments, the mammalian subject is a non-human primate, rodent (e.g., mouse or rat), bovine, porcine, equine, or livestock animal. In some embodiments, the HSCs are obtained from a human subject, such as a human subject with a hematopoietic malignancy. In some embodiments, the HSCs are obtained from a healthy donor. In some embodiments, the HSCs are obtained from a subject to whom immune cells expressing a chimeric receptor are subsequently administered. HSCs administered to the same subject from whom the cells were obtained are referred to as autologous cells, whereas HSCs obtained from a subject other than the subject to whom the cells are administered are referred to as allogeneic cells.

[0147] In some embodiments, the population of genetically engineered cells is a heterogeneous population of cells, e.g., a heterogeneous population of genetically engineered cells that contain different CD34 mutations. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of the gene encoding CD34 in the population of genetically engineered cells contain a mutation that is affected by the genome editing approach described herein, e.g., by the CRISPR / Cas system using the gRNA provided herein. As an example, the population of genetically engineered cells can contain a plurality of different CD34 mutations, and each of the plurality of mutations can contribute to the percentage of copies of CD34 in the population of cells that have the mutation.

[0148] In some embodiments, expression of CD34 on genetically engineered hematopoietic cells (e.g., HSCs) is compared to expression of CD34 on native hematopoietic cells (e.g., wild-type counterparts), e.g., native HSCs. In some embodiments, the genetic engineering results in a reduction in expression levels of CD34 by at least 50%, at least 60%, 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%, compared to expression of CD34 on native hematopoietic cells (e.g., wild-type counterparts). For example, in some embodiments, the genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD34 compared to native hematopoietic cells (e.g., wild-type counterparts).

[0149] In some embodiments, the genetic engineering described herein, e.g., using a gRNA targeting CD34 described herein, results in a reduction in expression levels of wild-type CD34 by at least 50%, at least 60%, 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%, as compared to the level of expression of wild-type CD34 on native hematopoietic cells (e.g., wild-type counterparts). For example, in some embodiments, the genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD34 compared to native hematopoietic cells (e.g., wild-type counterparts).

[0150] In some embodiments, the genetic engineering described herein, e.g., using a gRNA targeting CD34 described herein, results in a reduction in expression levels of a wild-type lineage-specific cell surface antigen (e.g., CD34) of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, by 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%, compared to a suitable control (e.g., a cell or cells). In some embodiments, a suitable control comprises a level of a wild-type lineage-specific cell surface antigen measured or predicted in a plurality of unmanipulated cells from the same subject. In some embodiments, a suitable control comprises a level of a wild-type lineage-specific cell surface antigen measured or predicted in a plurality of cells from a healthy subject. In some embodiments, a suitable control comprises a level of a wild-type lineage-specific cell surface antigen measured or predicted in a population of cells from a pool of healthy individuals (e.g., 10, 20, 50, or 100 individuals). In some embodiments, a suitable control includes the level of wild-type lineage-specific cell surface antigen measured or predicted in a subject in need of a treatment described herein, e.g., anti-CD34 treatment, e.g., where the subject has cancer and where the cells of the cancer express CD34.

[0151] In some embodiments, the methods of genetically engineering a cell described herein include providing a wild-type cell, e.g., a wild-type hematopoietic stem or progenitor cell. In some embodiments, a wild-type cell is an unedited cell that includes (e.g., expresses) two functional copies of a gene encoding CD34. In some embodiments, the cell includes a CD34 gene sequence according to SEQ ID NO: 113. In some embodiments, the cell includes a CD34 gene sequence that encodes a CD34 protein encoded in SEQ ID NO: 17 or 18, e.g., the CD34 gene sequence may include one or more silent mutations relative to SEQ ID NO: 17 or 18. In some embodiments, the cell used in the method is a natural cell or a non-engineered cell. In some embodiments, wild-type cells express CD34 or give rise to more differentiated cells that express CD34 at levels similar to (or within 90%-110%, 80%-120%, 70%-130%, 60%-140%, or 50%-150%) of a cell line that expresses CD34.

[0152] In some embodiments, wild-type cells give rise to more differentiated cells that bind antibodies that bind CD34 (e.g., anti-CD34 antibodies) or that bind such antibodies at levels similar to (or within 90%-110%, 80%-120%, 70%-130%, 60%-140%, or 50%-150%) antibody binding to cell lines expressing CD34, e.g., L1236, L428, KM-H2, and L591. Antibody binding may be measured, for example, by flow cytometry or immunohistochemistry.

[0153] In some embodiments, the CD34-edited cells provided herein are CD34-edited hematopoietic stem cells (HSCs). In some embodiments, such CD34-edited hematopoietic stem cells are functionally equivalent to non-edited (e.g., native) HSCs, e.g., in that they are capable of long-term engraftment in a recipient, e.g., in a clinical setting, of differentiating into all hematopoietic lineages, and of reconstituting the recipient's hematopoietic system, e.g., after hematopoietic stem cell transplantation. In some embodiments, the CD34-edited HSCs provided herein express cell surface markers characteristic of HSCs, except for CD34. Some suitable cell surface markers associated with hematopoietic stem cells are provided herein, and other suitable surface markers associated with hematopoietic stem cells will be apparent to one of skill in the art based on this disclosure. Suitable HSC markers include, for example, those disclosed in BD Biosciences Human and Mouse CD Marker Handbook, accessible at www.bd.com / documents / bd-legacy / catalogue / biosciences / DS_Human-Mouse-CD-Maker-Biosciences_CT_DE.pdf, last accessed December 30, 2021. Additional suitable HSC markers include, for example, those disclosed in Tomellini et al., Cell Reports 2019 28(4):1063-1073 (PMID:31340144 DOI:10.1016 / j.celrep.2019.06.084), the entire contents of each of which are incorporated herein by reference. Some exemplary suitable HSC markers include, but are not limited to, CD49c, CD71, CD90, CD117, CD135, CD243, CD292, CDw293, CD318, CD325, CD349, CD201, CD228, and CD309. In some embodiments, suitable HSC surface markers include the absence of surface markers characteristic for differentiated cells.For example, in some embodiments, CD34-edited HSCs provided herein, e.g., CD34-edited HSCs that are functionally indistinguishable from non-edited (e.g., native) HSCs, are negative for CD34 (CD34-) and express one or more surface markers characteristic of or typically expressed by native HSCs, e.g., native HSCs capable of long-term engraftment into a suitable recipient. In some embodiments, CD34-edited HSCs as provided herein are CD34- and express CD90 (CD90+), e.g., as determined by immunostaining through any other suitable method. In some embodiments, CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD71+. In some embodiments, CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD117+. In some embodiments, CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD135+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD243+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD292+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CDw293+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD318+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD325+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD349+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD201+. In some embodiments, CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD228+.In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD309+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD71+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD49c+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD201+. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and negative for any lineage markers associated with differentiated hematopoietic cells (lin-). In some embodiments, the lineage markers include CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, or CD235a, or any combination of two or more of these markers. Thus, in some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and negative for CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, or CD235a, or any combination of two or more of these markers. In some embodiments, the CD34-edited HSCs as provided herein are negative for CD34 (CD34-) and CD45RA-. In some embodiments, the CD34-edited HSCs as provided herein meet a combination of two or more of the criteria provided above. For example, in some embodiments, such CD34-edited HSCs are CD34-, CD90+, and CD71+. In some embodiments, such CD34-edited HSCs are CD34-, CD90+, CD71+, and lin-. In some embodiments, such CD34-edited HSCs are CD34-, CD90+, CD45RA-. In some embodiments, such CD34-edited HSCs are CD34-, CD90+, CD45RA-, and CD201+.In some embodiments, such CD34 edited HSCs are CD34-, CD90+, CD45RA-, and CD49c+; in some embodiments, such CD34 edited HSCs are CD34-, CD90+, CD45RA-, CD201+, and CD49c+. In some embodiments, such CD34 edited HSCs are CD34-, CD90+, CD45RA-, CD201+, CD49c+, and lin-. In some embodiments, the engineered hematopoietic stem cells do not express CD34 on their cell surface, and express CD90, CD201, and CD49c. In some embodiments, the engineered hematopoietic stem cells do not express CD34 on their cell surface, and express CD90, CD201, and CD49c, and do not express CD45c. In some embodiments, the genetically engineered hematopoietic stem cells do not express CD34 on their cell surface, express one or more of CD90, CD201, and CD49c, and are negative for CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD45RA-, CD56, CD123, and CD235a.

[0154] In some embodiments, the CD34-edited HSCs provided herein comprise a gene edit in a gene encoding CD34, resulting in a CD34 gene product being expressed on the surface of the CD34-edited HSC, e.g., an edited gene that does not encode a CD34 protein, or that encodes a CD34 protein that cannot be bound by a CD34-targeting antibody (e.g., has a modification in an epitope bound by a CD34-targeting antibody). In some embodiments, the CD34 gene editing comprises an indel resulting from NHEJ-mediated repair of a double stranded break in the gene encoding CD34, e.g., from an RNA-guided nuclease, which in turn results in premature termination and thus truncation of the gene product encoded by CD34, or results in an edited CD34 gene that does not otherwise encode a CD34 gene product expressed on the surface of the CD34-edited HSCs, e.g., a CD34 protein, or that encodes a CD34 protein that cannot be bound by a CD34-targeting antibody (e.g., has a modification in an epitope bound by a CD34-targeting antibody). In some embodiments, the CD34 gene editing includes a single nucleotide change, e.g., from base editing or prime editing, or due to HDR-mediated repair of a double-stranded break in the gene encoding CD34, which then results in premature termination and thus truncation of the gene product encoded by CD34, or results in an edited gene that does not otherwise encode a CD34 gene product, e.g., a CD34 protein, expressed on the surface of the CD34-edited HSC, or that encodes a CD34 protein that cannot be bound by a CD34-targeting antibody (e.g., has a modification in an epitope bound by a CD34-targeting antibody). In some embodiments, the CD34 gene editing results in a frameshift or splice variant. In some embodiments, the CD34 gene editing results in a deletion, in whole or in part, or an alteration of an epitope bound by a CD34 antibody.In some embodiments, the CD34 edited HSCs are not recognized by CD34-targeted antibodies, CD34-binding antibody fragments, or other immunobinding agents, such as CD34-binding scFvs, CD34-binding CARs, or CD34-binding antibody-drug conjugates. In some embodiments, the CD34 edited HSCs are not recognized by CD34-targeted immunotherapeutics, such as CD34-targeted CAR-T cells or CD34-targeted CAR-NK cells.

[0155] Dual gRNA Compositions and Uses Thereof In some embodiments, a gRNA provided herein (e.g., a gRNA provided in Table 1 or Table 2) can be used in combination with a second gRNA, e.g., to target a CRISPR / Cas nuclease to two sites in a genome. For example, in some embodiments, it may be desirable to produce hematopoietic cells that are deficient for CD34 and a lineage-specific cell surface antigen, e.g., CD33, CD123, CLL-1, CD19, CD30, CD5, CD6, CD7, CD38, or BCMA, and the cells can be resistant to two agents: an anti-CD34 agent and an agent that targets the lineage-specific cell surface antigen. In some embodiments, the hematopoietic cells are deficient for CD34 and a lymphocyte-specific cell surface antigen or a myeloid-specific cell surface antigen. In some embodiments, it is desirable to contact the cells with two different gRNAs that target different sites in CD34, e.g., to create two cleavages and create a deletion or insertion between the two cleavage sites. Thus, the present disclosure provides various combinations of gRNA and associated CRISPR system, and cells made by genome editing methods using such combinations of gRNA and associated CRISPR system.In some embodiments, CD34 gRNA binds to a different nuclease than the second gRNA.For example, in some embodiments, CD34 gRNA can bind to Cas9, and the second gRNA can bind to Cas12a, or vice versa.

[0156] In some embodiments, the first gRNA is a CD34 gene provided herein. and a second gRNA targeting a lineage-specific cell surface antigen selected from: BCMA, CD19, CD20, CD30, ROR1, B7H6, B7H3, CD23, CD33, CD38, C-type lectin-like molecule 1, CS1, IL-5, L1-CAM, PSCA, PSMA, CD138, CD133, CD70, CD5, CD6, CD7, CD13, NKG2D, NKG2D ligand, CLEC12A, CD11, CD123, CD56, CD30, CD14, CD66b, CD41, CD61, CD62, CD235a, CD146, CD326, LMP2, CD22, CD52, CD10, CD3 / TCR, CD79 / BCR, and CD26.

[0157] In some embodiments, the first gRNA is a CD34 gRNA provided herein (e.g., a gRNA provided in Table 1 or Table 2 or a variant thereof) and the second gRNA targets a lymphocyte-specific cell surface antigen (e.g., a T cell, B cell, or NK cell specific cell surface antigen). In some embodiments, the lymphocyte-specific cell surface antigen is CD3, CD4, CD8, CD19, CD20, or CD56. In some embodiments, the first gRNA is a CD34 gRNA provided herein (e.g., a gRNA provided in Table 1 or Table 2 or a variant thereof) and the second gRNA targets a myeloid-specific cell surface antigen (e.g., a monocyte, macrophage, neutrophil, basophil, dendritic cell, erythrocyte, or platelet specific cell surface antigen). In some embodiments, the myeloid-specific cell surface antigen is CD11c, CD123, CD14, CD33, CD66b, CD41, CD61, CD62, or CD235a. Additional lymphoid and myeloid-specific cell surface antigens, and the cell types with which they are associated, can be found, for example, in the BD Biosciences Human and Mouse CD Marker Handbook, available at www.bd.com / documents / bd-legacy / catalogue / biosciences / DS_Human-Mouse-CD-Maker-Biosciences_CT_DE.pdf, last accessed on December 30, 2021, which list is incorporated herein by reference.

[0158] In some embodiments, the first gRNA is a CD34 gRNA provided herein (e.g., a gRNA provided in Table 1 or Table 2 or a variant thereof) and the second gRNA is a gRNA that is specific for the treatment of a neoplastic or malignant disease or disorder, such as a particular type of cancer, including, but not limited to, CD20, CD22 (non-Hodgkin's lymphoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (acute myeloid leukemia (AML)), CD10 (gp100) (common (pre-B) acute lymphocytic leukemia and The antibodies target lineage-specific cell surface antigens associated with CD3 / T cell receptor (TCR) (T cell lymphoma and leukemia), CD79 / B cell receptor (BCR) (B cell lymphoma and leukemia), CD26 (epithelial and lymphoid malignancies), human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (lymphoid malignancies), RCAS1 (gynecologic, biliary, and pancreatic ductal adenocarcinomas), and prostate-specific membrane antigen.

[0159] In some embodiments, the first gRNA is a CD34 gRNA provided herein (e.g., a gRNA provided in Table 1 or Table 2, or a variant thereof) and the second gRNA is a gRNA selected from the group consisting of CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD2 7, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA , CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD5 9, CD60a, CD61, CD62E, CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66F, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD7 5S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85A, CD85C, CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD85J, CD85K, CD86, CD87, CD88, C D89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108 , CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD121a, CD121b, CD122, CD123,<h2 style=";text-align:left;direction:ltr">CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD 135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD1 4、CDw145、CD146、CD147、CD148、CD150、CD152、CD152、CD153、CD154、CD155 、CD156a、CD156b、CD156c、CD157、CD158b1、CD158b2、CD158d、CD158e1 / e2、C D158f、CD158g、CD158h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、C D161、CD163、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD1 72a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、C D179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD 192、CD193、CD194、CD195、CD196、CD197、CDw198、CDw199、CD200、CD201、CD 202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210a、CDw210b、 CD212, CD213a1, CD213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232 CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, C D254, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD272, CD272, CD273, CD274, CD275, CD276, CD277CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a , CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD312, CD314, CD315, CD316, CD317 , CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362, or CD363.

[0160] In some embodiments, the second gRNA is a gRNA disclosed in WO2017 / 066760, WO2019 / 046285, WO / 2018 / 160768, or Borot et al. PNAS (2019) 116(24):11978-11987, each of which is incorporated by reference in its entirety.

[0161] Method of administration to a subject in need thereof Some embodiments of the present disclosure provide methods comprising administering to a subject in need thereof an effective number of genetically engineered cells as described herein that contain modifications in their genome that result in loss of expression of CD34, or expression of a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34.

[0162] A subject in need thereof, in some embodiments, is a subject undergoing or about to undergo immunotherapy targeting CD34. A subject in need thereof, in some embodiments, is a subject having or diagnosed with an autoimmune disease characterized, for example, by adverse immune activity of CD34-expressing cells. A subject in need thereof, in some embodiments, is a subject having or diagnosed with a malignancy characterized by expression of CD34 on malignant cells. In some embodiments, a subject having such a malignancy or autoimmune disease may be a candidate for immunotherapy targeting CD34, but the risk of adverse on-target, off-disease effects may outweigh the expected or observed benefit to the subject. In some such embodiments, administration of the engineered cells described herein results in amelioration of adverse on-target, off-disease effects because the engineered cells provided herein are not efficiently targeted by immunotherapeutic agents targeting CD34.

[0163] Examples of autoimmune diseases for which the cells, compositions, and methods described herein may be useful include, but are not limited to, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune disease of the inner ear (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy (AMA). N), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis (EoE) , eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, polyangiogranulomatosis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid of pregnancy (PG), hidradenitis suppurativa (HS) (acne inverse), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM) , Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes mellitus (type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, Chronic Lyme disease, Meniere's disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multifocal motor neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neonatal lupus optica, Neutropenia, Ocular mucous membrane pemphigoid, Optic neuritis,Relapsing rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy -, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis-giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, or Vogt-Koyanagi-Harada disease.

[0164] In some embodiments, subjects with such malignancies or autoimmune diseases are candidates for radiation therapy, e.g., to ablate malignant cells (e.g., malignant cells expressing CD34). In some embodiments, the risk of adverse off-target effects (e.g., on adjacent or surrounding cells or tissues) and on-target off-disease effects (e.g., on non-malignant CD34-expressing cells) may outweigh the expected or observed benefits to the subject of radiation therapy. In some embodiments, administration of engineered cells (e.g., engineered hematopoietic cells, e.g., HSCs) described herein after radiation therapy results in amelioration of adverse on-target, off-disease effects. Without wishing to be bound by theory, the combination of immunotherapeutic approaches, including lymphocytic effector cells targeting CD34, such as CAR-T cells or CAR-NK cells, and genetically engineered cells (e.g., genetically engineered stem cells, e.g., HSCs) that do not express CD34, express reduced levels of CD34 (e.g., compared to wild-type cells), or express variant forms of CD34 that are not recognized by CD34-targeting immunotherapeutic agents, is believed to be an alternative to radiation therapy for subjects with CD34-expressing malignancies or autoimmune diseases characterized by deleterious immune activity of CD34-expressing cells. Immunotherapeutic approaches targeting CD34 are believed to avoid or significantly reduce the risk of off-target effects (e.g., on adjacent or surrounding cells or tissues). Replenishment of depleted stem cell populations with immunotherapy-resistant engineered cells (e.g., engineered stem cells, e.g., HSCs) that do not express CD34, express reduced levels of CD34 (e.g., compared to wild-type cells), or express variant forms of CD34 that are not recognized by immunotherapeutic agents that target CD34, is believed to ameliorate or eliminate the on-target off-disease effects of immunotherapeutic approaches.

[0165] In some embodiments, the malignancy is a hematological malignancy, or cancer of the blood. In some embodiments, the malignancy is a lymphoid malignancy. Generally, lymphoid malignancies are associated with the inappropriate production, development, and / or function of lymphatic cells, such as T-lineage or B-lineage lymphocytes. In some embodiments, the malignancy is characterized by or associated with cells that express CD34 on the cell surface.

[0166] In some embodiments, the malignancy is associated with abnormal T lymphocytes, such as a T lineage cancer, for example, a T cell leukemia or a T cell lymphoma.

[0167] Examples of T-cell leukemias and T-cell lymphomas include, but are not limited to, T-lineage acute lymphoblastic leukemia (T-ALL), Hodgkin's lymphoma or non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), large granular lymphocytic leukemia, adult T-cell leukemia / lymphoma (ATLL), T-cell prolymphocytic leukemia (T-PLL), T-cell chronic lymphocytic leukemia, T-prolymphocytic leukemia, T-cell lymphocytic leukemia, and T-cell lymphoma. lymphoma, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), enteropathy-associated T-cell lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic lymphoma, anaplastic large cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic gamma delta T-cell lymphoma, lymphoblastic lymphoma, or hairy cell leukemia.

[0168] In some embodiments, the malignancy is associated with abnormal B lymphocytes, such as a B lineage cancer, such as a B cell leukemia or B cell lymphoma, In some embodiments, the malignancy is B lineage acute lymphoblastic leukemia (B-ALL) or chronic lymphocytic leukemia (B-CLL), primary mediastinal B cell lymphoma.

[0169] In some embodiments, cells of the malignant tumor express CD34, for example, on their surface.

[0170] In some embodiments, the malignant tumor comprises a population of cells characterized by expression of CD34. In some embodiments, the population of cells characterized by expression of CD34 are cancer stem cells. Without wishing to be bound by theory, the cancer stem cell theory suggests that for some malignant tumors, cancer stem cells share many properties with normal, healthy stem cells. In some embodiments, the cancer stem cells express CD34, for example, on their surface. In some embodiments, the immunotherapeutic approaches described herein include, for example, lymphocytic effector cells, such as CAR-T cells or CAR-NK cells, that target CD34 to specifically target the cancer stem cells of the malignant tumor. In some embodiments, the immunotherapeutic approaches described herein that target cancer stem cells also have on-target off-disease effects, for example, detrimental to healthy stem cells. In some embodiments, genetically engineered cells (e.g., genetically engineered stem cells, e.g., HSCs) that do not express CD34, express reduced levels of CD34 (e.g., compared to wild-type cells), or express a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34, are used to replenish or replace non-cancer stem cells (e.g., healthy stem cells) that are targeted by an immunotherapeutic approach.

[0171] In some embodiments, the malignancy is graft-versus-host disease.

[0172] Also within the scope of the present disclosure are malignancies that are considered relapsed and / or refractory, such as relapsed or refractory hematological malignancies. A subject in need thereof, in some embodiments, is a subject who is undergoing or will undergo immune effector cell therapy, e.g., CAR-T cell therapy, that targets CD34, in which the immune effector cells express a CAR that targets CD34, and in which at least a subset of the immune effector cells also express CD34 on their cell surface, or in which healthy stem cells (e.g., HSCs) in the subject undergoing therapy express CD34 on their cell surface. As used herein, the term "fratricide" refers to self-killing. For example, cells of a population of cells kill or induce the killing of cells of the same population. In some embodiments, cells of the immune effector cell therapy kill or induce the killing of other cells of the immune effector cell therapy. In such embodiments, fratricide ablates a portion or the entire population of immune effector cells before a desired clinical outcome can be achieved, e.g., ablation of malignant cells expressing CD34 in a subject. In some such embodiments, engineered immune effector cells may be used to avoid such fratricide and the associated negative impact on treatment outcome, as provided herein, for example, immune effector cells that do not express CD34 or do not express a CD34 variant recognized by a CAR, as immune effector cells that form the basis of immune effector cell therapy. In such embodiments, engineered immune effector cells may be further modified to also express a CD34-targeted CAR, as provided herein, for example, immune effector cells that do not express CD34 or do not express a CD34 variant recognized by a CAR. In some embodiments, the immune effector cells may be lymphocytes, e.g., T lymphocytes, e.g., alpha / beta T lymphocytes, gamma / delta T lymphocytes, or natural killer T cells, etc. In some embodiments, the immune effector cells may be natural killer (NK) cells.

[0173] In some embodiments, the cells of the immune effector cell therapy kill or induce the killing of stem cells (e.g., HSCs) in the subject that express CD34 on their cell surface. In some embodiments, the methods described herein result in depletion of a targeted stem cell niche (e.g., HSC niche) in the subject. In some embodiments, the methods described herein do not change or appreciably change the level or viability of stem cells in at least one non-targeted stem cell niche in the subject. In some embodiments, the methods described herein target all stem cell niches of a particular type in the subject (e.g., all HSC niches). In some embodiments, the methods described herein result in complete depletion of a stem cell niche (e.g., HSC niche) in the subject. As used herein, stem cell niche refers to an anatomical region of a subject that contains a specific microenvironment that contains a population of stem cells in an undifferentiated, self-renewing state.

[0174] In some embodiments, the supply of stem cells (e.g., HSCs) in a subject is replenished by administering to the subject engineered stem cells that do not express CD34 or that express a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34. In some embodiments, the subject is administered engineered stem cells that do not express CD34 or that express a variant form of CD34 that is not recognized by immunotherapeutic agents that target CD34 in combination with immune effector cells that target CD34 (e.g., engineered immune effector cells provided herein, such as immune effector cells that do not express CD34 or that do not express a CD34 variant recognized by a CAR, which may also be further modified to express a CD34-targeting CAR).

[0175] In some embodiments, an effective number of engineered cells as described herein contain modifications in their genome that result in loss of expression of CD34 or expression of a variant form of CD34 that is not recognized by an immunotherapeutic agent that targets CD34, and are administered to a subject in need thereof, e.g., a subject undergoing or who will undergo an immunotherapy that targets CD34, where the immunotherapy is associated, or at risk of being associated, with deleterious on-target, off-disease effects, e.g., in the form of cytotoxicity against healthy cells in the subject that express CD34. In some embodiments, an effective number of such engineered cells may be administered to a subject in combination with an anti-CD34 immunotherapeutic agent.

[0176] It is understood that when agents (e.g., CD34 modified cells and an anti-CD34 immunotherapeutic agent) are administered in combination, the cells and agent can be administered at the same time or at different times, e.g., in close temporal proximity. Further, the cells and agent can be mixed or mixed in separate volumes or dosage forms. For example, in some embodiments, administration in combination includes administration during the same course of treatment, e.g., during the course of treating a subject with an anti-CD34 immunotherapy, where an effective number of engineered, CD34 modified cells can be administered to a subject simultaneously or sequentially with the anti-CD34 immunotherapy, e.g., before, during, or after treatment.

[0177] In some embodiments, an immunotherapeutic agent that targets CD34 as described herein is an immune cell that expresses a chimeric antigen receptor that comprises an antigen-binding fragment (e.g., a single chain antibody) capable of binding to CD34. The immune cell can be, for example, a T cell (e.g., a CD4+ or CD8+ T cell) or a NK cell.

[0178] A chimeric antigen receptor (CAR) may comprise a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain, such as one derived from a stimulatory molecule. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule, such as 4-1BB (i.e., CD137), CD27, and / or CD28, or fragments of those molecules. The extracellular antigen binding domain of the CAR may comprise a CD34-binding antibody fragment. The antibody fragment may comprise one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or any combination of the foregoing.

[0179] Chimeric antigen receptors (CARs) typically comprise an antigen binding domain, e.g., an antibody fragment, fused to a CAR framework, which may include a hinge region (e.g., from CD8 or CD28), a transmembrane domain (e.g., from CD8 or CD28), one or more costimulatory domains (e.g., CD28 or 4-1BB), and a signaling domain (e.g., from CD3 zeta). Exemplary sequences of CAR domains and components are provided, for example, in PCT Publication WO2019 / 178382 and in Table 3 below. Table 3: Exemplary components of chimeric antigen receptors [Table 3]

[0180] In some embodiments, the number of genetically engineered cells, e.g., HSCs, HPCs, or immune effector cells, provided herein, administered to a subject in need thereof is greater than or equal to 10 6 ~10 11 However, amounts below or above this exemplary range are also within the scope of the present disclosure. For example, in some embodiments, the number of genetically engineered cells provided herein, e.g., HSCs, HPCs, or immune effector cells, administered to a subject in need thereof is about 10 6 , about 10 7 , about 10 8, about 10 9 , about 10 10 , or about 10 11 In some embodiments, the number of genetically engineered cells provided herein, e.g., HSCs, HPCs, or immune effector cells, administered to a subject in need thereof is 10 6 ~10 9 Within the range of 10 6 ~10 8 Within the range of 10 7 ~10 9 Within the range of about 10 7 ~10 10 Within the range of 10 8 ~10 10 Within the range of 10 9 ~10 11 is within the range.

[0181] In some embodiments, the immunotherapeutic agent targeting CD34 is an antibody-drug conjugate (ADC). An ADC may be a molecule comprising an antibody or an antigen-binding fragment thereof conjugated to a toxin or drug molecule. Binding of the antibody or fragment thereof to the corresponding antigen allows delivery of the toxin or drug molecule to a cell (e.g., a target cell) that displays the antigen on its cell surface, thereby resulting in the death of the target cell.

[0182] Suitable antibodies and antibody fragments that bind CD34 that can be used, for example, for the generation of chimeric antigen receptors, immunotherapy of CAR-T cells expressing such chimeric receptors, or for the generation of antibody-drug conjugates will be apparent to those skilled in the art. Examples of suitable anti-CD34 antibodies include, but are not limited to, EP373Y (Abcam (rabbit, unconjugated)), clone QBEnd-10 (LifeSpan BioSciences (mouse)), clone MEC14.7 (LifeSpan BioSciences (rat)), clone SI16-01 (antibody name ET1606-11; HUABIO (rabbit), clone HPCA1 / 2598R (biorbyt (rabbit), 4C8 and humanized h4C8, 4H11, cQBEND / 10 or hQBEND / 10 variants of QBEnd-10, 27H2, CIRMA-K4, DS554AB, 10C304, 9C5, B-C34, RM300, AC136.

[0183] Any suitable antibodies and antigen-binding fragments capable of binding human CD34 (mRNA NCBI Reference Sequence: NM_001025109.1, protein NCBI Reference Sequence: NP_001020280.1) can be used in conjunction with the compositions and methods described herein. There are two isoforms of CD34 that differ in the length of their cytoplasmic tails (long and short). Recently, stable cell lines expressing CD34 have been generated using the long isoform and can be used as an immunogen (see, e.g., Adv. Pharm. Bull. 5:69-75, 2015). CD34, such as the long isoform of CD34, can be used as an immunogen to identify antibodies and antigen-binding fragments thereof capable of binding CD34 and generate suitable CD34-targeted immunotherapies for the treatment of cancer and autoimmune diseases, such as CD34-targeted CAR and CAR-T cells, or antibody-drug conjugates, as well as for use as pre-treatment agents before hematopoietic stem cell transplantation therapy.

[0184] Some suitable CD34 antibodies, CD34-binding antibody fragments, and CD34-binding scFvs are described herein, and additional suitable CD34 antibodies, CD34-binding antibody fragments, and CD34-binding scFvs will be apparent to one of skill in the art based on this disclosure, including, but not limited to, the CD34-binding antibodies produced and released from ATCC Accession Nos. AC133.1 and HB 12346, for example, as described in U.S. Patent No. 5,843,633, which is incorporated herein by reference.

[0185] Suitable CD34 binding antibodies and CD34 binding antibody fragments and scFvs that may be used in conjunction with the compositions and methods described herein further include humanized variants of CD34 antibodies, CD34 binding antibody fragments, scFvs, and any antibodies, antibody fragments, and scFvs that specifically bind to the same CD34 epitopes as described herein, as assessed, for example, by competitive CD34 binding assays.

[0186] Suitable toxins or drugs for use in antibody-drug conjugates are known in the art and will be apparent to one of ordinary skill in the art (see, e.g., Peters et al. Biosci. Rep. (2015) 35(4):e00225; Beck et al. Nature Reviews Drug Discovery (2017) 16:315-337; Marin-Acevedo et al. J. Hematol. Oncol. (2018) 11:8; Elgundi et al. Advanced Drug Delivery Reviews (2017) 122:2-19).

[0187] In some embodiments, the antibody-drug conjugate may further comprise a linker (e.g., a peptide linker, such as a cleavable linker) attaching the antibody and the drug molecule.

[0188] Examples of suitable toxins or drugs for antibody-drug conjugates include, but are not limited to, brentuximab vedotin, glembatumumab vedotin / CDX-011, depatukizumab mafodotin / ABT-414, PSMA ADC, polatuzumab vedotin / RG7596 / DCDS4501A, denintuzumab mafodotin / SGN-CD19A, AGS-16C3F, CDX-014, RG7841 / DLYE5953A, RG7882 / DMUC406A, RG7986 / DCDS0780A, SGN-LIV1A, enfortumab vedotin / ASG-22ME, AG-15ME, AGS67E, terisotuzumab vedotin / ABBV-399, ABBV-221, ABBV-085, GSK-2857916, tisotuzumab vedotin / HuMax-TF-ADC, HuMax-Axl-ADC, pinatuzumab vedotin / RG7593 / DCDT2980S, rifatuzumab vedotin / RG7599 / DNIB0600A, indusatumab vedotin / MLN-0264 / TAK-264, bundletuzumab vedotin / RG7450 / DSTP3086S, sofituzumab vedotin / RG7458 / DMUC5754A, RG7600 / DMOT4039A, RG7336 / DEDN6526A, ME1547, PF-06263507 / ADC 5T4, trastuzumab emtansine / T-DM1, mirvetuximab soravtansine / IMGN853, cortuzumab emtansine / SAR3419, naratuximab emtansine / IMGN529, indatuximab emtansine / BT-062, anetumab emtansine / BAY 94-9343, SAR408701, SAR428926, AMG 224, PCA062, HKT288, LY3076226, SAR566658, lorvotuzumab mertansine / IMGN901, cantuzumab mertansine / SB-408075, cantuzumab mertansine / IMGN242, laprituximab Emtansine / IMGN289, IMGN388, bivatuzumab mertansine, AVE9633, BIIB015, MLN2704, AMG 172, AMG 595, LOP 628, vadatuximabTaliline / SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-CD352A, rovalpituzumab tesirin / SC16LD6.5, SC-002, SC-003, ADCT-301 / HuMax-TAC-PBD, ADCT-402, MEDI3726 / ADC-401, IMGN779, IMGN632, gemtuzumab ozogamicin, inotuzumab ozogamicin / CMC-544, PF-06647263, CMD-193, CMB-401, trastuzumab duocarmazine / SYD985, BMS-936561 / MDX-1203, sacituzumab govitecan / IMMU-132, labetuzumab govitecan / IMMU-130, DS-8201a, U3-1402, milatuzumab doxorubicin / IMMU-110 / hLL1-DOX, BMS-986148, RC48-ADC / hertuzumab-vc-MMAE, PF-06647020, PF-06650808, PF-06664178 / RN927C, rupartumab amidotin / This includes the toxins and drugs contained in BAY1129980, aprtumab ixadotin / BAY1187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276, and DSTA4637S / RG7861.

[0189] In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell surface lineage-specific protein can induce internalization of the antibody-drug conjugate, releasing the drug (or toxin) intracellularly. In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell surface lineage-specific protein can induce internalization of the toxin or drug, thereby allowing the toxin or drug to kill a cell expressing the lineage-specific protein (target cell). In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell surface lineage-specific protein can induce internalization of the toxin or drug, thereby allowing the toxin or drug to modulate the activity of a cell expressing the lineage-specific protein (target cell). The type of toxin or drug used in the antibody-drug conjugates described herein is not limited to any particular type.

[0190] General Technology The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press; Culture(RIFreshney,ed.1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.1993-8)J.Wiley and Sons;Methods in Enzymology(Academic Press,Inc.):Handbook of Experimental Immunology (DMWeir and CCBlackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987; Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984); Animal Cell Culture (R.I. Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986); and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.) are fully described in...

[0191] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are therefore to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0192] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0193] The present disclosure is further illustrated by the following examples, which are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way. EXAMPLES

[0194] Example 1: Gene editing of CD34 in human cells Design of sgRNA constructs

[0195] The target domains and gRNAs shown in Tables 1 and 2 were designed by manual inspection for PAM sequences for applicable nucleases, e.g., Cas9, proximal to the target region and prioritized according to predicted specificity by minimizing potential off-target sites in the human genome using online search algorithms (e.g., Benchling algorithm, Donench et al. 2016, Hsu et al. 2013). Figures 1 and 2A-2C show the location of exemplary gRNAs within the CD34 gene. All designed synthetic sgRNAs were produced with chemically modified nucleotides at the three terminal positions at both the 5' and 3' ends. The modified nucleotides included 2'-O-methyl-3'-phosphorothioates (abbreviated as "ms") and the ms-sgRNAs were HPLC purified.

[0196] Editing human CD34+ HSCs

[0197] Frozen CD34+ HSCs were thawed according to the manufacturer's instructions. To edit HSCs, -HSCs were thawed and cultured in StemSpan SFEM medium supplemented with StemSpan CC110 cocktail (StemCell Technologies) for approximately 48 hours prior to electroporation with RNP, as shown in Figure 3A. To electroporate HSCs, approximately 2 x 10 5 The cells were pelleted, resuspended in electroporation solution, and mixed with 3 μg Cas9 and 3 μg gRNA in the form of RNP. CD34+ HSCs were electroporated using standard electroporation procedures. The experimental conditions are provided in Table 4.

[0198] Table 4. [Table 4]

[0199] Genomic DNA analysis

[0200] Approximately 48 hours after electroporation, cells were collected for cell count, viability analysis, purification, and sequencing. For all genomic analyses, DNA was collected from cells, amplified with primers flanking the target region, purified, and allele modification frequency was analyzed using TIDE (tracking indels by resolution). Analysis was performed using a reference sequence from a mock-transfected sample.

[0201] Human CD34+ cells were electroporated with Cas9 protein and presented with CD34-targeting gRNA as described above. Editing rates were determined by % indels as assessed by TIDE analysis. Editing efficiency was determined by TIDE analysis as shown in Figure 3D and Figure 4.

[0202] Flow cytometry analysis

[0203] CD34 gRNA-edited cells may also be evaluated for surface expression of CD34 protein, for example, by flow cytometry analysis (FACS). Live HSCs are stained for CD34 using anti-CD34 antibody and analyzed by flow cytometry on an Attune NxT flow cytometer (Life Technologies). Cells genetically modified with the CD34 gene show a reduction in CD34 expression as detected by FACS.

[0204] Viability of edited cells

[0205] At 24 and 48 hours after ex vivo editing, the average cell number and percentage of viable edited CD34KO and control cells were quantified using flow cytometry and 7AAD viability dye. As shown in Figures 3B and 3C, CD34KO cells edited using the CD34 gRNA described herein were viable and remained viable over time after electroporation and gene editing, similar to that observed in control cell editing with gRNAs targeting CD5, mock-edited cells, and non-electroporated cells.

[0206] Example 2: Large scale electroporation - 96 hour time point Frozen CD34+ HSCs were thawed according to the manufacturer's instructions. To edit HSCs, ~HSCs were thawed and cultured in StemSpan SFEM medium supplemented with StemSpan CC110 cocktail (StemCell Technologies) for approximately 48 hours prior to electroporation with RNP, as shown in Figure 5A. To electroporate HSCs, approximately 1 x 10 6 The cells were pelleted, resuspended in electroporation solution, and mixed with 15 μg Cas9 and 15 μg gRNA in the form of RNPs. CD34+ HSCs were electroporated using standard electroporation procedures. The experimental conditions are provided in Table 5.

[0207] Table 5. [Table 5]

[0208] At 24 and 96 hours after electroporation, cells were collected for cell counts, viability analysis, purification, and sequencing analysis. The cell counts and viability of cells edited using the indicated CD34 gRNAs were found to have similar levels of viability as mock-edited cells (Mock) or cells that were not electroporated (No EP), as shown in Figures 5B and 5C.

[0209] CD34 gRNA edited cells were also assessed for surface expression of CD34 protein by flow cytometry analysis (FACS) using an anti-CD34 antibody (anti-CD34-PE Texas Red). Cells genetically modified for the CD34 gene were found to have reduced CD34 expression as detected by FACS. See Figures 6A, 6B, and 7A-7D.

[0210] Example 3: Large scale electroporation - 168 hour time point Frozen CD34+ HSCs were thawed according to the manufacturer's instructions. To edit HSCs, ~HSCs were thawed and cultured in StemSpan SFEM medium supplemented with StemSpan CC110 cocktail (StemCell Technologies) for approximately 48 hours prior to electroporation with RNP, as shown in Figure 8. To electroporate HSCs, ~1 x 10 6 The cells were pelleted, resuspended in electroporation solution, mixed with 15 μg Cas9 and 15 μg gRNA in the form of RNPs, and then electroporated using standard electroporation procedures. The experimental conditions are provided in Table 6.

[0211] Table 6. [Table 6]

[0212] At 24, 48, 120, 144, and 168 hours after electroporation, cells were collected for cell counts, viability analysis, purification, and sequencing analysis. Cell counts and viability of cells edited using the indicated CD34 gRNAs were found to have similar viability to mock-edited cells (Mock) or cells that were not electroporated (No EP).

[0213] CD34 gRNA-edited cells were also assessed for surface expression of CD34 protein by flow cytometry analysis (FACS) using an anti-CD34 antibody (anti-CD34-PE Texas Red). At 48 hours after electroporation, cells genetically modified with the CD34 gene were found to have slightly reduced CD34 expression as detected by FACS. See Figures 9A and 9B. However, at 168 hours after electroporation, cells genetically modified with the CD34 gene were found to have reduced CD34 expression as detected by FACS. See Figures 10A and 10B.

[0214] Example 4: CAR-T cytotoxicity assay Genetically modified cells produced using the gRNAs shown in Tables 1 or 2 may be assessed for killing by CD34-CAR T cells.

[0215] CAR constructs and lentivirus production

[0216] The second generation CAR is constructed to target CD34. An exemplary CAR construct consists of an extracellular scFv antigen-binding domain using the CD8α signal peptide, CD8α hinge and transmembrane region, 4-1BB costimulatory domain, and CD3ξ signaling domain. The anti-CD34 scFv sequence can be obtained from any anti-CD34 antibody known in the art. The CAR cDNA sequence for the target is subcloned into the multiple cloning site of the pCDH-EF1α-MCS-T2A-GFP expression vector, and lentivirus is generated according to the manufacturer's protocol (System Biosciences). Lentivirus can be generated by transient transfection of 293TN cells (System Biosciences) using Lipofectamine 3000 (ThermoFisher). An exemplary CAR construct is generated by cloning the light and heavy chains of an anti-CD34 antibody into the CD8α hinge domain, ICOS transmembrane domain, ICOS signaling domain, 4-1BB signaling domain, and CD3ξ signaling domain into the lentiviral plasmid pHIV-Zsgreen.

[0217] CAR transduction and expansion

[0218] Human primary T cells are isolated from Leuko Pak (Stem Cell Technologies) by magnetic bead separation using anti-CD4 and anti-CD8 microbeads according to the manufacturer's protocol (Stem Cell Technologies). Purified CD4+ and CD8+ T cells are mixed 1:1 and activated using anti-CD3 / CD28-conjugated Dynabeads (Thermo Fisher) at a bead-to-cell ratio of 1:1. The T cell culture medium used is CTS Optimizer T cell growth medium supplemented with Immune Serum Replacement, L-Glutamine and GlutaMAX (all purchased from Thermo Fisher) and 100 IU / mL IL-2 (Peprotech). T cell transduction is performed 24 hours after activation by spinoculation in the presence of polybrene (Sigma). CAR-T cells are cultured for 9 days before cryopreservation. Before all experiments, T cells are thawed and rested at 37°C for 4-6 hours.

[0219] Flow cytometry-based CAR-T cytotoxicity assay

[0220] The cytotoxicity of the target cells is measured by comparing the viability of the target cells to the viability of the negative control cells. For the CD34 cytotoxicity assay, CD34+ cells may be used as target cells, and CD34+ cells deficient in CD34 or with reduced expression of CD34 may be generated as described in Examples 1-3.

[0221] Target cells and negative control cells are stained with CellTrace Violet (CTV) and CFSE (Thermo Fisher), respectively, according to the manufacturer's instructions. After staining, target cells and negative control cells are mixed 1:1.

[0222] Anti-CD34 CAR-T cells are used as effector T cells. Non-transduced T cells (mock CAR-T) are used as control. Effector T cells are co-cultured with target cell / negative control cell mixture in duplicate at an effector to target ratio of 1:1. A group of target cell / negative control cell mixture alone without effector T cells is included as a control. Cells are incubated at 37°C for 24 hours before flow cytometry analysis. Propidium iodide (ThermoFisher) is used as a viability dye. For calculation of specific cell lysis, the fraction of live target cells relative to live negative control cells (referred to as target fraction) is used. Specific cell lysis is calculated as (target fraction without effector cells - target fraction with effector cells) / (target fraction without effector) x 100%.

[0223] Example 5: Effect of anti-CD34 antibody drug conjugates on engineered HSCs Genetically modified cells produced using the gRNAs shown in Table X may be assessed for killing by antibody-drug conjugates, such as anti-CD34 antibodies to immunotoxins.

[0224] Frozen CD34+ HSPCs derived from mobilized peripheral blood were thawed and cultured for 72 hours prior to electroporation with ribonucleoprotein containing Cas9 and sgRNA. Samples were electroporated under the following conditions: i. Mash (Cas9 only), and ii. KO sgRNA (such as any one of the CD34 gRNAs shown in Table X). Cells are allowed to recover for 72 hours and genomic DNA is harvested and analyzed. The percentage of CD34 positive cells is assessed by flow cytometry to confirm that editing with CD34 gRNA is effective in knocking out or reducing CD34 expression. Editing events in HSCs result in various indel sequences.

[0225] Sensitivity of cells with CD34 deletions to antibody-drug conjugates

[0226] To determine in vitro toxicity, cells are incubated with the antibody-drug conjugate in culture medium and the number of viable cells is quantified over time. Engineered cells deficient in CD34 or with reduced CD34 expression generated with the CD34 gRNA described herein are more resistant to antibody-drug conjugate treatment than cells expressing full-length CD34 (mock).

[0227] (ii) Enrichment of CD34-edited cells

[0228] To assay whether CD34 modified cells are enriched after treatment with antibody-drug conjugates, HSPCs are edited with a standard nuclease (e.g., Cas9, Cpf1) to gRNA ratio of 50%. The bulk population of cells is analyzed before and after treatment with the antibody-drug conjugates. After treatment with the antibody-drug conjugates, CD34 edited cells are enriched, such that the percentage of CD34-deficient cells is increased.

[0229] (iii) In vitro differentiation of CD34-edited HSPCs

[0230] Cell populations are assessed for lymphoid differentiation before and after treatment with antibody-drug conjugates at various days after differentiation. Engineered CD34 knockout cells generated using the CD34 gRNAs described herein may show increased expression of differentiation markers, whereas cells expressing full-length CD34 (mock) may not differentiate.

[0231] Example 6: Assessment of persistence of CD34KO cells in vivo. Editing in mobilized peripheral blood CD34+HSCs (mPB CD34+HSPCs).

[0232] The gRNA (Synthego) was designed as described in Examples 1-3. mPB CD34+ HSPCs were purchased from Fred Hutchinson Cancer Center and thawed according to the manufacturer's instructions. These cells were then edited via CRISPR / Cas9 as described in Examples 1-3 using the CD34-targeting gRNA described herein, as well as a non-CD34-targeting control gRNA (gCtrl) designed not to target any region in the human or mouse genome.

[0233] At 4, 24, and 48 hours after ex vivo editing, the percentage of viable edited CD34KO and control cells is quantified using flow cytometry and 7AAD viability dye. High levels of CD34KO cells edited using the CD34 gRNAs described herein are viable and can remain viable for hours after electroporation and gene editing, comparable to that observed in control cells edited with a non-CD34-targeting control gRNA, gCtrl.

[0234] In addition, to determine the editing percentage as assessed by indels (insertion / deletion) 48 hours after ex vivo editing, genomic DNA is collected from cells, PCR amplified with primers flanking the target region, purified, and analyzed by TIDE as described in Examples 1-3.

[0235] After TIDE analysis, the percentage of long-term HSCs (LT-HSCs) after editing with the CD34 gRNA described herein is quantified by flow cytometry. The percentage of LT-HSCs after editing with the specified CD34 gRNA is evaluated. This assay can be performed upon cryopreservation of edited cells before injection into mice, for example, to study the persistence of CD34KO cells in vivo. Edited cells are diluted to 5×10 in a medium volume of 1 mL per vial. 6 Cells / mL are cryopreserved in CS10 medium (Stem Cell Technology).

[0236] The engraftment efficiency and persistence of CD34KO mPB HSPCs in vivo will be investigated.

[0237] Female NSG mice (JAX), 6-8 weeks of age, are allowed to acclimate for 2-7 days. After acclimation, the mice are irradiated using 175 cGy total body irradiation with an X-ray irradiator. This is considered day 0 of the study. 4-10 hours after irradiation, the mice are engrafted with CD34KO cells generated during any of the CD34 gRNAs described herein or control cells edited with gCtrl. Cryopreserved cells are thawed and counted using a BioRad TC-20 automated cell counter. The number of viable cells is quantified in the thawed vial, which is used to prepare the total number of cells for engraftment in the mice. Mice are engrafted with 1 x 10 cells in a 100 μL volume. 6 A single intravenous injection of edited cells is given. Body weights and clinical observations are recorded weekly for each mouse in the four groups.

[0238] At 8 and 12 weeks after engraftment, 50 μL of blood is collected from each mouse by retro-orbital bleeding for analysis by flow cytometry. At 16 weeks after engraftment, mice are sacrificed and blood, spleen, and bone marrow are collected for analysis by flow cytometry. Bone marrow is isolated from femurs and tibias. Bone marrow from femurs is also used for on-target editing analysis. Flow cytometry is performed using FACSCanto™ 10 color and BDFACSDiva™ software. Cells are generally first sorted by viability using 7AAD viability dye (live / dead analysis), and then live cells are gated by expression of human CD45 (hCD45), but not mouse CD45 (mCD45). Cells that are hCD45+ are then further gated for expression of human CD19 (hCD19) (lymphoid cells, specifically B cells). Cells expressing human CD45 (hCD45) are also gated and analyzed for the presence of various cell markers of the myeloid lineage.

[0239] The number of cells expressing each of the analyzed markers, being comparable across all mice regardless of which edited cells were engrafted, indicates successful engraftment of CD34 gRNA-edited CD34KO cells in the blood of the mice.

[0240] At 8, 12, and 16 weeks post-engraftment, the percentage of nucleated blood cells that are hCD45+ is quantified in groups of mice (n=15 mice / group) that received control cells edited with control gRNA (gCtrl) or CD34KO cells by dividing the absolute hCD45+ cell count by the absolute mouse CD45+ (mCD45) cell count.

[0241] The percentage of hCD34+ cells in the blood will also be quantified at week 8 post-engraftment in control and CD34KO mouse groups. Mice engrafted with CD34KO cells (edited with any of the CD34 gRNAs described herein) are expected to have significantly lower levels of hCD34+ cells compared to mice engrafted with control cells at weeks 8, 12, and 16.

[0242] Next, the percentages of specific populations of differentiated cells in the blood, such as CD19+ lymphoid cells, hCD14+ monocytes, and hCD11b+ granulocytes / neutrophils, are quantified at 8, 12, and 16 weeks post-engraftment in mice engrafted with CD34KO or control cells. Levels of hCD19+, hCD14+, and hCD11b+ cells in the blood were comparable between the control and CD34KO groups, and the levels of these cells remained comparable from 8 to 16 weeks post-engraftment. Comparable levels of hCD19+, hCD14+, and hCD11b+ cells in the blood indicate that similar levels of human myeloid and lymphoid cell populations are present in mice receiving CD34KO cells and mice receiving control cells.

[0243] Finally, amplicon-seq can be performed on bone marrow samples isolated 16 weeks post-engraftment to analyze on-target CD34 editing in mice engrafted with edited CD34KO cells.

[0244] Cell samples obtained from the spleens of engrafted animals are evaluated.

[0245] At 16 weeks post-engraftment, the percentages of hCD45+ cells and hCD34+ cells are also quantified in the spleens of mice engrafted with control or CD34KO cells. Comparable levels of hCD45+ cells and reduced levels of hCD34+ cells between groups of mice (engrafted with control or CD34KO cells) may indicate long-term persistence of CD34KO HSCs in the spleens of NSG mice.

[0246] In addition, the percentages of hCD14+ monocytes, hCD11b+ granulocytes / neutrophils, CD19+ lymphoid cells, and hCD3+ T cells in the spleen are quantified 16 weeks after engraftment. Comparable levels of hCD14+, hCD11b+, hCD19+, and hCD3+ cells in the spleen between control and CD34KO groups may indicate that the edited CD34KO cells are capable of multilineage human hematopoietic cell reconstitution in the spleen of NSG mice.

[0247] Blood and bone marrow neutrophils are assessed.

[0248] Sixteen weeks after engraftment, the percentage of hCD11b+ cells is quantified in the blood and bone marrow of mice engrafted with control or CD34KO cells. Comparable levels of CD11b+ neutrophil populations were observed in both the blood and bone marrow of NSG mice engrafted with control and CD34KO cells, indicating successful engraftment and differentiation.

[0249] Blood and bone marrow myeloid and lymphoid progenitor cells are assessed.

[0250] Also, the percentage of hCD123+ cells in the blood and bone marrow, as well as the percentage of hCD10+ cells in the bone marrow, will be quantified in mice engrafted with control or CD34KO cells at week 16. Comparable levels of myeloid and lymphoid progenitor cells between the control and CD34KO groups may indicate successful engraftment and development.

[0251] Example 7: Genotyping of CD34-edited HSC colony forming units (CFUs). This example demonstrates the characterization of exemplary CD34 editing by CFU and indel analysis prior to differentiation. HSCs were edited using exemplary guide RNAs: CD34-2 or CD34-3.

[0252] FIG. 11A shows the experimental approach used for HSC editing in this and subsequent examples. Briefly, 2×10 6 Frozen CD34 + HSCs were thawed and cultured in StemSpan SFEM medium supplemented with StemSpan CC110 cocktail (StemCell Technologies) for approximately 48 hours after electroporation with RNP. Approximately 2 × 10 5 2×10 cells were pelleted, resuspended in electroporation solution, and mixed with 3 μg Cas9 and 3 μg gRNA in the form of RNP. CD34+ HSCs were electroporated using standard electroporation procedures. 5 Cells were included in the samples for editing (electroporated with RNPs containing CD34-2 and Cas9, or with RNPs containing CD34-3 and Cas9), as well as a no-electroporation (no EP) control, and a sample electroporated with a control guide (RNP containing a control guide RNA designed not to target any region in the human genome).

[0253] Assessment of edited CD34 HSC CFU

[0254] CFU levels were similar in mock-treated edited HSCs and HSCs (Figures 11B-11C). These results suggest that CD34 editing does not affect cell differentiation. TIDE analysis was used to determine editing efficiency in individual CD34-edited BFUs and CFUs obtained using CD34-2 (guide 2) and CD34-3 (guide 3). Appropriate editing efficiency was observed in the majority of analyzed BFUs and CFUs (Figures 11D and 11E). Analysis of edits generated in CD34-edited HSCs showed that +1 indels were predominant (72%) in cells edited using CD34-2, resulting in a frameshift leading to premature termination of the transcript (Figures 11F, 11L, 11N, and 11P). The predominance of indels produced by editing using CD34-2 thus leads to the production of truncated proteins. CD34-3 edited cells had a predominant -12 indel (32%) resulting in a deletion of four amino acids, followed by a -1 indel (24%) resulting in a frameshift leading to premature termination of the transcript (Figures 11G, 11O, and 11Q).

[0255] BFUs and CFUs were further analyzed to determine whether the edits were monoallelic or biallelic. Analysis of edits generated in CD34-edited BFUs and CFUs obtained using guides CD34-2 and CD34-3 showed a high percentage of biallelic edits (Figures 11H-11K). Biallelic edits were observed for the two guides examined in both CFUs and BFUs (Figures 11J and 11K). Indel analysis in individual colonies confirmed a predominant +1 indel for guide CD34-2 and predominant -12 and -1 indels for guide CD34-3 (Figures 11L-11O). Analysis of the predominant indel species for both guides showed that +1 indels for guide CD34-2 lead to premature termination and truncation (Figure 11N), while the predominant -12 indel for guide CD34-3 leads to a deletion of four amino acids in the extracellular domain of CD34, and the next most frequent -1 indel for this guide leads to premature termination and truncation (Figure 11Q).

[0256] CD34 knockout time course study

[0257] The viability of three different donor HSC samples (donor 1, donor 2, and donor 3) was assessed after CD34 editing using guide RNA CD34-2 or CD34-3. Cells were prepared and electroporated as described above. At 24 and 48 hours after ex vivo editing, the average cell number and percentage of live CD34-edited cells (using guides CD34-2 and CD34-3) and control cells (not electroporated (no EP) or not electroporated using a control guide that does not target CD34 (sgCTRL)) were quantified. Although CD34-edited cells were viable and remained viable over time after electroporation and gene editing, no significant differences in viability were observed in CD34-edited cells compared to control cells electroporated with control gRNA and cells that were not electroporated (Figures 12A-12C).

[0258] For all genomic analyses, DNA was collected from cells, amplified with primers flanking the target region, purified, and allele modification frequency was analyzed using TIDE (tracking indels by degradation). ICE analysis of edited cells using both "no EP" and "sgCTRL" datasets as references showed that indel generation was detectable at 48 hours and remained present at 216 hours in all three edited donor HSC samples edited with either CD34-2 guide (Figures 12D, 12F, and 12H) or CD34-3 guide (Figures 12E, 12G, and 12I). The predominance of indels present in edited donor HSCs corresponds to those observed in the previous part of the examples.

[0259] A decrease in surface CD34 expression in edited HSCs was detected by flow cytometry in some cases as early as 48 hours after electroporation, and in all donor samples at 120 hours after electroporation (Figures 12J-12V). These results confirm the successful disruption of CD34 in three donor samples of HSCs using either one of the exemplary guides CD34-2 or CD34-3. The percentage of CD34+ cells at different time points was calculated for samples edited using guides CD34-2 or CD34-3, as well as for the "no EP" and "sgCTRL" control samples, averaged together across two different donors (Figure 12W). The results and calculated standard deviations show that editing with the two exemplary guides across donors significantly reduced the frequency of CD34+ cells, producing populations with approximately 85% of cells showing no detectable CD34 expression 216 hours after electroporation with CD34-2, and greater than 70% of cells showing no detectable CD34 expression 216 hours after electroporation with CD34-3.

[0260] Example 8: In vitro differentiation (IVD) of CD34 myeloid cells. This example demonstrates the characterization of in vitro differentiation capacity of CD34-edited human HSPCs after editing using the exemplary guide RNAs provided herein. Cell populations were assessed for myeloid differentiation upon CRISPR / Cas9 editing of CD34 using guides CD34-2 or CD34-3 at various days after differentiation, which was achieved by culturing cells in granulocytic and monocytic induction medium conditions. The experimental approach followed is shown in Figure 13A.

[0261] Assessing editing outcomes in in vitro differentiated HSCs

[0262] 48 hours after electroporation, the presence of edited genomic DNA in cells cultured in IVD medium was confirmed by PCR amplification followed by gel electrophoresis (Figure 13B). TIDE analysis of DNA collected from edited HSCs 48 hours after electroporation determined the editing efficiency of indel generation at +1 position in CD34-2 treated cells and at -1 and -12 positions in CD34-3 treated cells, confirming successful editing results (Figures 13C and 13D). Immunophenotypic analysis of differentiated cells arising from CD34-edited HSCs demonstrated that CD34 editing did not affect the differentiation potential of CD34-edited cells compared to no EP and gCTRL control HSCs (Figures 13E-13L). No significant differences in the expression of differentiation markers were observed between CD34-edited and control cell populations. For example, expression of the monocyte surface marker CD11b or CD14 or CD15 granulocyte marker over time, assessed as % surface marker positive cells or by geometric mean fluorescence intensity, was not significantly different in the CD34-edited cell population compared to the control cell population. These results demonstrate that CD34 editing does not destroy the differentiation capacity of edited HSCs to differentiate into the myeloid lineage or induce a differentiation bias.

[0263] Example 9: Characterization of CD34 edited mice in vivo. In this example, in vivo experiments are described to characterize CD34-edited HSCs in vivo in a mouse model. An exemplary experimental setup is shown in Figure 14A.

[0264] Briefly, human CD34+ HSCs were edited via CRISPR / Cas9 as described herein using exemplary CD34-targeting guide RNAs (CD34-2 and CD34-3), as well as a non-CD34-targeting control gRNA (gCtrl). 24 and 48 hours after ex vivo editing, the percentage of live CD34-edited and control cells was quantified, and appropriate viability (>80%) and editing (>60%) levels were confirmed as described in more detail elsewhere herein. Edited cells were cultured at 5×10 in CS10 medium (Stem Cell Technology). 6 Cells / mL were cryopreserved in a medium volume of 1 mL per vial.

[0265] Cryopreserved cells were thawed and counted using a BioRad TC-20 automated cell counter. The number of viable cells was quantified in the thawed vial and used to prepare the total number of cells for engraftment in mice. On day 0 of the experiment, female NSG mice (JAX) aged 6-8 weeks were irradiated using 175 cGy total body irradiation, and 4-10 hours after irradiation, mice were engrafted with CD34 edited cells generated as described herein or control cells electroporated with gCtrl. Mice were engrafted with 1 × 10 6 cells in a 100 μL volume. 6 Each mouse was given a single intravenous injection of the edited cells. Body weights and clinical observations were recorded weekly for each mouse in the three groups.

[0266] At 8, 9, and 12 weeks post-engraftment, 50 μL of blood is collected from each mouse by retro-orbital bleeding for analysis by flow cytometry. At 16 weeks post-engraftment, mice are sacrificed and blood, spleen, bone marrow, and thymus are collected for analysis by flow cytometry. Bone marrow is isolated from femurs and tibias. Bone marrow from femurs is also used for on-target editing analysis. Flow cytometry is performed using Beckman Coulter CytoFLEX LX software. Cells are generally first sorted for viability (e.g., using 7AAD viability dye (live / dead analysis)), and then live cells are gated by expression of human CD45 (hCD45), but not mouse CD45 (mCD45). Cells that are hCD45+ are then further gated for expression of human lymphoid or myeloid cell surface markers to demonstrate engraftment and repopulation of the blood system with cells derived from human HSCs. For example, cells expressing human CD45 are analyzed for expression of CD19 (hCD19) to detect lymphoid cells, specifically B cells, or for expression of other lymphoid cell markers, such as T cell markers or NK cell markers. Cells expressing human CD45 (hCD45) are also analyzed for the presence of various cell markers of the myeloid lineage.

[0267] At 8, 12, and 16 weeks post-engraftment, the percentage of nucleated blood cells that are hCD45+ is quantified in groups of mice (n=10 mice / group) that received control cells edited with control gRNA (gCtrl) or CD34-edited cells. This value is calculated by dividing the absolute hCD45+ cell count by the absolute mouse CD45+ (mCD45) cell count.

[0268] The percentage of hCD34+ cells in the blood is also quantified at 8 weeks post-engraftment in control and CD34-edited mouse groups as an assessment for human and mouse cell chimerism. Mice engrafted with CD34-edited cells (e.g., edited with CD34-2 as described herein) have similar levels of hCD34+ cells compared to mice engrafted with control cells (not CD34 edited, e.g., no electroporation, or edited using a control guide that does not target CD34) at weeks 8, 12, and 16.

[0269] The percentages of specific populations of differentiated cells are then analyzed. For example, the percentages of CD19+ lymphoid cells, hCD14+ monocytes, and hCD11b+ granulocytes / neutrophils are quantified at 8, 12, and 16 weeks post-engraftment in mice engrafted with CD34-edited or control cells. The levels of hCD19+, hCD14+, and hCD11b+ cells in the blood are comparable between the control and CD34-edited groups, and the levels of these cells remain comparable from 8 to 16 weeks post-engraftment. Comparable levels of hCD19+, hCD14+, and hCD11b+ cells in the blood are observed, indicating successful engraftment and differentiation of CD34-edited HSCs in irradiated NSG mice, demonstrating that similar levels of human myeloid and lymphoid cell populations are present in mice receiving CD34-edited cells and mice receiving control cells.

[0270] The presence and frequency of CD34 editing is confirmed in engrafted cells in NSG animals that received the CD34-edited cell population. Amplicon-seq is performed on bone marrow samples isolated 16 weeks post-engraftment to analyze CD34 editing in mice engrafted with edited CD34-edited cells. The presence of CD34-edited cells in bone marrow is confirmed, demonstrating the ability of CD34-edited cells to successfully engraft. The presence of CD34-edited HSC is confirmed by immunophenotyping of bone marrow-derived cells or circulating PBMC cells and identifying CD34-edited cells that are CD34-, CD45RA-, CD49c+, CD90+, CD201+, and lin-.

[0271] At 16 weeks post-engraftment, the percentage of hCD11b+ cells is quantified in the blood and bone marrow of mice engrafted with control or CD34-edited cells. Comparable levels of CD11b+ neutrophil populations observed in mice engrafted with control cells and CD34-edited cells in both the blood and bone marrow of NSG mice indicate successful engraftment and differentiation.

[0272] At 16 weeks after engraftment, the percentages of hCD45+ cells and hCD34+ cells are also quantified in the spleens of mice engrafted with control or CD34-edited cells. Comparable levels of hCD45+ cells and reduced levels of hCD34+ cells between groups of mice (engrafted with control or CD34-edited cells) may indicate long-term persistence of CD34-edited HSCs in the spleens of NSG mice. In addition, at 16 weeks after engraftment, the percentages of hCD14+ monocytes, hCD11b+ granulocytes / neutrophils, CD19+ lymphoid cells, and hCD3+ T cells in the spleen are quantified. Comparable levels of hCD14+ cells, hCD11b+ cells, hCD19+ cells, and hCD3+ cells in the spleen between the control and CD34-edited groups may indicate that CD34-edited cells are capable of multilineage human hematopoietic cell reconstitution in the spleen of NSG mice.

[0273] At 16 weeks post-engraftment, the percentage of hCD45+ cells and the percentage of hCD34+ cells are also quantified in the thymus of mice engrafted with control or CD34-edited cells. In addition, the percentage of CD3+ cells (as a percent of hCD45+ cells) in the thymus is quantified at 16 weeks post-engraftment. Comparable levels of hCD3+ in the thymus between the control and CD34-edited groups may indicate that the CD34-edited cells are capable of multilineage reconstitution of human hematopoietic cells in the thymus of NSG mice.

[0274] Example 10: Ablation of CD34+ HSCs in subjects with hematopoietic disorders. Subjects with hematopoietic disorders, e.g., hematological malignancies in which malignant cells (e.g., cancer stem cells) express CD34, are treated with allogeneic hematopoietic cell transplant (HCT) including CD34-edited HSCs and with anti-CD34 immunotherapeutics, e.g., a CD34 antibody conjugated to a calicheamicin moiety.

[0275] For HCT, a population of cells comprising CD34-edited hematopoietic stem cells is obtained according to the methods described herein, for example, by obtaining HSCs from a healthy donor that is HLA matched at the 8 / 8 locus (HLA-A, -B, -C, DRB1) to the subject, and editing the HSCs using guide CD34-2 or CD34-3.

[0276] HSCs are obtained from donors after mobilization with G-CSF / Plerixa in up to two apheresis procedures. A minimum of 10 x 10 6 Viable cells / kg (where kg refers to the body weight of the recipient subject) are obtained from the donor by apheresis for processing, editing, and subsequent administration to the recipient subject. From this apheresis product, at least 3.0×10 6 Viable cells / kg of recipient weight undergo minimal manipulation and are cryopreserved to serve as a backup stem cell source, e.g., for use as a rescue dose. The remainder of the apheresis product is used for processing and preparation of CD34-edited populations for HCT.

[0277] CD34-edited HSC populations for HCT are prepared by enriching the apheresis product for CD34+ cells, followed by electroporation and editing with the CD34 gRNA / Cas9 complex, as described above, using the CD34 guides CD34-2 or CD34-3 as described herein.

[0278] The CD34-edited cells are then placed in culture for approximately 48 hours. At harvest, after the culture period is completed, the cells are washed, resuspended in the final formulation, and cryopreserved. Cell viability and editing efficiency are confirmed using a representative sample, and CD34-edited HSC populations confirmed to contain at least 70% live cells and at least 45% CD34 editing efficiency are used for HCT. CD34-edited cell populations for administration to subjects meet these viability and editing efficiency criteria and contain at least 3×10 6 The recipient subject's CD34-edited HSC population comprises at least 4×10 cells / kg body weight of the recipient subject. 6 cells / kg, 5×10 6 cells / kg, 6×10 6 cells / kg, or 7 x 10 6 Contains cells / kg.

[0279] The subject is conditioned using routine clinical procedures to ablate the subject's hematopoietic stem cells, including any malignant cells that express CD34.After completion of the conditioning regimen, the subject undergoes HCT via intravenous (IV) infusion with thawed CD34-edited HSCs.The day of HCT is day 0 of the treatment regimen.

[0280] Subjects are assessed for engraftment of CD34-edited HSCs on day 28 by measuring the absolute peripheral neutrophil count (ANC) for CD34-edited (CD34-, e.g., truncated CD34 or epitope-edited CD34) neutrophils in the subject. A subject is considered to exhibit neutrophil recovery (also referred to as successful CD34-edited neutrophil engraftment) if the subject exhibits an absolute peripheral CD34-edited neutrophil count of > 1000 / dL CD33- ANC on day 28 post-HCT.

[0281] If the subject shows neutrophil recovery on day 28, a bone marrow biopsy is obtained from the subject on day 60 to assess disease status and hematopoietic recovery. The percentage of donor chimerism and CD34-edited myelopoiesis is also determined from peripheral blood at this time. If the subject shows successful CD34-edited HSC engraftment and CD34-edited hematopoiesis on day 60, the subject is then administered a CD34 antibody-drug conjugate. Administration of a CD34-targeted immunotherapeutic agent, e.g., an anti-CD34 antibody-drug conjugate, is preferably initiated within 30 days of the bone marrow biopsy on day 60, i.e., preferably by day 90. However, initiation of the CD34-targeted immunotherapeutic agent may be delayed until day 120, if the subject's clinical condition requires such a delay in order to allow the subject to achieve a CD33- ANC of >= 1000 / dL, e.g., in light of comorbidities, including HCT-related comorbidities. If a CD34-targeted immunotherapy is initiated more than 30 days after the day 60 bone marrow biopsy, a repeat bone marrow biopsy is completed prior to initiating the CD34-targeted immunotherapy.

[0282] The CD34-targeting immunotherapeutic drug is administered at its recommended dose to ablate all CD34-expressing hematopoietic cells in the subject.However, some subjects may be administered a lower dose, for example, in the event of treatment-related adverse effects, for example, dose-limiting toxicity (DLT), or taking into account the health status, comorbidities, or medical history of each individual subject.

[0283] Alternatively, the subject is not subjected to a conventional conditioning regimen to ablate the subject's own CD34-expressing blood cells prior to HCT, but the subject's CD34-expressing cells are targeted by administration of a CD34-targeted immunotherapeutic agent, such as a CD34-targeted antibody-drug conjugate or a CD34-targeted CAR-T cell population. In such a scenario, the subject may be administered a cycle of immunotherapeutic agent prior to HCT of the CD34-edited HSCs, and a cycle of immunotherapeutic agent after HCT. Alternatively, the subject may be administered an immunotherapeutic agent in one or more cycles following HCT, thus completely preceding the pre-HCT conditioning regimen.

[0284] After administration of immunotherapy, the subject is evaluated for the presence of malignant cells.A significant reduction in the number of malignant cells, particularly CD34-expressing malignant cells, or complete ablation of CD34-expressing malignant cells is observed in the subject.In addition, the long-term engraftment of CD34-edited HSCs and the reconstitution of all cell lineages from CD34-edited HSCs are observed in the subject.

[0285] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0286] Articles such as "a", "an", and "the" can mean one or more, unless indicated to the contrary or otherwise clear from the context. A claim or description containing "or" between two or more members of a group is considered to be satisfied when one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise clear from the context. The disclosure of a group containing "or" between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which one or more members of the group are present, and embodiments in which all of the members of the group are present. For purposes of brevity, those embodiments are not individually set forth herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0287] It should be understood that the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, or descriptive terms from one or more claims or one or more relevant parts of the description are introduced into another claim. For example, a claim that is dependent on another claim may be amended to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, if a claim describes a composition, it should be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein, or, in some cases, according to methods known in the art, are included, unless otherwise indicated or unless it is clear to one skilled in the art that a contradiction or inconsistency may arise.

[0288] When elements are presented as a list, for example in Markush group format, it should be understood that all possible subgroups of the elements are also disclosed, and that any element or subgroup of elements can be excluded from the group. It should also be noted that the term "comprising" is intended to be open, permitting the inclusion of additional elements or steps. In general, when an embodiment, product, or method is referred to as comprising a particular element, feature, or step, it should be understood that an embodiment, product, or method consisting of or consisting essentially of such element, feature, or step is also provided. For purposes of brevity, those embodiments are not individually set forth herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0289] When ranges are given, the endpoints are included. Moreover, unless otherwise indicated or clear from the context and / or understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume any particular value within the stated range, in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values ​​in each range are not individually set forth herein, but it will be understood that each of these values ​​is provided herein and can be specifically claimed or disclaimed. Unless otherwise indicated or clear from the context and / or understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any subrange within the given range, in which the endpoints of the subrange are expressed to the same precision as the tenth of the unit of the lower limit of the range.

[0290] It should also be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. When a range is given, any value within the range may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods described herein may be excluded from any one or more of the claims. For the purpose of brevity, not all of the embodiments in which one or more elements, features, objects, or aspects are excluded are explicitly set forth herein.

Claims

1. A genetically engineered hematopoietic cell, or its progeny, that contains a modified gene encoding CD34.

2. (a) the modified gene encoding CD34 contains an indel mutation; (b) the modified gene encoding CD34 is modified such that an exon is skipped; (c) exon 1, exon 2, or exon 3 of the modified gene encoding CD34 has been genetically engineered; (d) the modified gene encoding CD34 contains an insertion or deletion generated by a non-homologous end joining (NHEJ) event or a homology directed repair (HDR) event; and / or (e) the modified gene encoding CD34 results in loss of function of CD34 in the genetically engineered hematopoietic cells, or their progeny; The hematopoietic cell or progeny thereof according to claim 1.

3. The hematopoietic cell or progeny thereof of claim 1 or 2, wherein the modified gene encoding CD34 contains an insertion or deletion immediately proximal to a site cleaved by an RNA-guided nuclease when coupled to a gRNA comprising a targeting domain provided by any one of SEQ ID NOs: 12, 13, 11, 14 or 15.

4. The hematopoietic cell of any one of claims 1 to 3, or a progeny thereof, wherein expression of CD34 is reduced or eliminated compared to a wild-type counterpart cell not carrying a modified gene encoding CD34, and optionally wherein expression of CD34 is less than 25% compared to the expression level of CD34 in a wild-type counterpart cell not carrying a modified gene encoding CD34.

5. A hematopoietic cell, or its progeny: (a) has reduced or no binding to an agent that comprises an anti-CD34 binding domain; (b) retain the ability to differentiate normally compared to a population of non-genetically engineered hematopoietic cells; and / or (c) lacking a CD34 epitope or having a modified CD34 epitope; A hematopoietic cell according to any one of claims 1 to 4, or a progeny thereof.

6. 6. The hematopoietic cell of any one of claims 1 to 5, or a progeny thereof, further comprising a modified gene encoding a lineage-specific cell surface antigen, optionally wherein the lineage-specific cell surface antigen is CD33, CD123, CLL-1, CD312, CD19, CD30, CD5, CD6, CD7, CD38, BCMA, or CD45.

7. The hematopoietic cell, or progeny thereof, of claim 6 , wherein the hematopoietic cell, or progeny thereof, has reduced or no binding to an agent comprising a binding domain that targets a lineage-specific cell surface antigen.

8. A guide RNA (gRNA) comprising a targeting domain, wherein the targeting domain comprises a sequence selected from the group consisting of SEQ ID NOs: 12, 13, 11, 14, and 15.

9. 9. The gRNA of claim 8, wherein the gRNA comprises a first complementarity domain, a linking domain, a second complementarity domain that is complementary to the first complementarity domain, and a proximal domain.

10. The gRNA of claim 8 or 9, wherein the gRNA is a single guide RNA (sgRNA).

11. The gRNA of any one of claims 8 to 10, wherein the gRNA comprises one or more chemically modified nucleotide residues, one or more nucleotide residues comprising a 2'O-methyl moiety, one or more nucleotide residues comprising a phosphorothioate, and / or one or more nucleotide residues comprising a ThioPACE moiety.

12. 1. A method for producing a genetically engineered cell, comprising: a. providing a cell; and b. contacting a cell with (i) a gRNA according to any one of claims 8 to 11, and (ii) an RNA-guided nuclease that binds the gRNA, whereby the gRNA of (i) forms and / or maintains a ribonucleoprotein (RNP) complex with the RNA-guided nuclease of (ii), and the RNP complex forms an RNP complex under conditions suitable for binding a target domain in the genome of the cell; The method.

13. 13. The method of claim 12, wherein the RNA-guided nuclease is a CRISPR / Cas nuclease, optionally wherein the CRISPR / Cas nuclease is a Cas9 nuclease, a spCas nuclease, a saCas nuclease, a Cpf1 nuclease, or a base editor.

14. (a) the contacting comprises introducing into the cell the gRNA of (i) and the RNA-guided nuclease of (ii) in the form of a preformed ribonucleoprotein (RNP) complex; (b) the contacting comprises introducing into the cell the gRNA of (i) and / or the RNA-guided nuclease of (ii) in the form of a nucleic acid encoding the gRNA of (i) and / or the RNA-guided nuclease of (ii), optionally wherein the nucleic acid is RNA, preferably an mRNA or an mRNA analog; and / or (c) the ribonucleoprotein complex is introduced into the cell via electroporation; 14. The method according to claim 12 or 13.

15. The method of any one of claims 12 to 14, wherein the cell is a hematopoietic cell, a hematopoietic stem cell, a hematopoietic progenitor cell, an immune effector cell, a lymphocyte, or a T-lymphocyte.

16. A genetically engineered cell, wherein the cell is obtained or obtainable by the method according to any one of claims 12 to 15.

17. A cell population comprising a genetically engineered cell according to any one of claims 1 to 11 and 16.

18. A pharmaceutical composition comprising a genetically engineered hematopoietic cell according to any one of claims 1 to 11 and 16, or a progeny thereof, or a cell population according to claim 17.

19. 20. The pharmaceutical composition of claim 18 for use in a method comprising administering the pharmaceutical composition to a subject in need thereof.

20. 20. The pharmaceutical composition of claim 19, further comprising administering to the subject a therapeutically effective amount of at least one agent that targets CD34, wherein the agent comprises an antigen-binding fragment that binds to CD34, and optionally wherein the agent is an antibody-drug conjugate or an immune effector cell expressing a chimeric antigen receptor (CAR).

21. If the subject is: (a) a disease associated with cells expressing CD34; (b) a malignancy associated with or characterized by expression of CD34 on malignant cells, optionally where the malignant cells are cancer stem cells; and / or (c) hematopoietic malignant tumor or autoimmune disease, 21. The pharmaceutical composition of claim 19 or 20, comprising:

22. A genetically engineered hematopoietic stem cell comprising a modification in the gene encoding CD34, wherein the genetically engineered hematopoietic stem cell does not express the native CD34 protein, and wherein the genetically engineered hematopoietic stem cell is functionally indistinguishable from a native hematopoietic stem cell that expresses CD34.

23. Genetically engineered hematopoietic stem cells: (a) expressing one or more hematopoietic stem cell markers; (b) expresses one or more of CD49c, CD71, CD90, CD117, CD135, CD201, CD228, CD243, CD292, CDw293, CD309, CD318, CD325, and CD349; (c) does not express CD34 on its cell surface; (d) does not express on its cell surface a CD34 epitope recognized by an anti-CD34 antibody; (e) do not express lineage-specific surface markers characteristic of differentiated hematopoietic cells (lin-); (f) does not express CD2, CD3, CD4, CD8, CDl lb, CD14, CD15, CD16, CD19, CD56, CD123, or CD235a, or any combination thereof; (g) capable of long-term engraftment in a human recipient; and / or (h) capable of reconstituting the hematopoietic system in a human recipient after engraftment; 23. The genetically engineered hematopoietic stem cell of claim 22.

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