Donor T cells with a kill switch

Abstract

The disclosed methods are generally directed to preventing, treating, suppressing, controlling, or otherwise mitigating adverse reactions of T cell therapies designed to accelerate immune reconstitution, induce graft-versus-malignant effects, and / or target tumor cells. In some embodiments, the present disclosure provides a delivery vehicle comprising components adapted to knock out HPRT. In some embodiments, the delivery vehicle comprises a gRNA molecule and an endonuclease, e.g., a Cas protein. In some embodiments, the gRNA molecule targets a location contained within exon 2, 3, or 8 of the HPRT1 gene.

Description

Technical field

[0001] Cross-reference to related applications This application claims the benefit of the filing date of US Provisional Application No. 63 / 044,697, filed June 26, 2020, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates generally to gene therapy, particularly hematopoietic stem cells and / or lymphocytes, such as T cells transduced by an expression vector. The disclosure also relates to gene editing, eg, by the CRISPR-Cas system. [Background technology]

[0003] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematologic malignancies and inherited disorders of blood cells such as sickle cell disease. A challenge associated with allo-HSCT involves identifying a suitable source of donor cells. Matched related donors (MRD) and matched unrelated donors (MUD) provide a source of HSCs with lower associated risks, but the availability of these donors is significantly reduced compared to the availability of haploidentical donors (typically parents or siblings), from whom almost everyone obtains readily. However, there are complications associated with the use of haploidentical donors for allo-HSCT, most importantly the potential development of graft-versus-host disease (GvHD), which remains an obstacle to the success of allo-HSCT. It is believed that approximately half of patients undergoing allo-HSCT will develop GvHD requiring treatment, and more than 10% of patients may die from it. GvHD presents a heterogeneous manifestation involving multiple organ systems including the gastrointestinal tract, skin, mucous membranes, liver and lungs. Immunosuppressants have served as a central strategy for preventing and reducing GvHD. Currently, standard treatment of GvHD with corticosteroids has had very limited success as many patients develop steroid-refractory disease. There is no clear consensus on the best second-line and third-line approaches in treating acute and chronic GvHD (see Non-Patent Document 1).

[0004] Haploidentical transplantation often depletes T cells to reduce the risk of developing GvHD. However, the lack of donor T cells can render the transplant recipient immunocompromised and lead to an increased rate of fatal infections in transplanted patients. Moreover, more recent studies have shown that the presence of donor T cells significantly improves donor cell engraftment, thereby reducing the potential need for repeat HSCT, in addition to providing T cell immunity for the extended time required for CD4+ and CD8+ T cell engraftment (up to 2 years).

[0005] In the allo-HSCT malignancy setting, the benefits conferred by the presence of donor T cells include anti-tumor activity, or graft-versus-tumor (GVT) effect (also known as graft-versus-leukemia-GVL). The first report of donor lymphocyte infusion (DLI) resulting in disease palliation after relapse after HSCT was in 1990 in a patient with chronic myelogenous leukemia (CML). Prior to DLI, patients who relapsed after HSCT appeared to have succumbed to their disease and few received a second transplant. After success in CML, DLI was then utilized in other hematologic malignancies such as acute leukemia and myeloma. A key challenge, therefore, relates to the proper balance of GVT effects, which are the factors that enable sustained alleviation, but also the toxicity associated with GvHD. [Prior art documents] [Non-Patent Literature]

[0006] [Non-Patent Document 1] Jamil, M.O. & Mineishi, S. Int J Hematol (2015) 101:452 [Outline of the invention] [Problems to be solved by the invention]

[0007] Gene therapy strategies that modify human stem cells hold great promise for the treatment of many human diseases. It is believed that the full therapeutic potential of allo-HSCT will not be realized until approaches are developed that minimize GvHD while simultaneously maintaining the positive contribution of donor T cells. [Means for solving the problem]

[0008] A first aspect of the present disclosure provides a method of providing the benefits of lymphocyte transfusion to a patient in need of such treatment while mitigating side reactions, comprising: (a) transfecting or transducing lymphocytes obtained from a donor sample with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in one of exons 3 or 8 of the HPRT1 gene to generate a population of lymphocytes substantially deficient in HPRT; (b) substantially deficient in HPRT. positively selecting the modified lymphocyte population ex vivo to provide an engineered lymphocyte population; and (c) administering a therapeutically effective amount of the engineered lymphocyte population to the patient. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments, the method further comprises administering the HSC graft to the patient. In some embodiments, the HSC graft is administered prior to administration of the modified lymphocyte population, concurrently with administration of the modified lymphocyte population, or after administration of the modified lymphocyte population.

[0009] In some embodiments, the guide RNA molecule targets a sequence contained in exon 3 of the HPRT1 gene. In some embodiments, the guide RNA molecule targets a sequence contained in exon 8 of the HPRT1 gene. In some embodiments, guide RNA molecules targeting sequences contained in one of exons 3 or 8 of the HPRT1 gene have at least 90% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 3 or 8 of the HPRT1 gene has at least 91% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 3 or 8 of the HPRT1 gene has at least 92% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 3 or 8 of the HPRT1 gene has at least 93% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, guide RNA molecules targeting sequences contained in one of exons 3 or 8 of the HPRT1 gene have at least 94% sequence identity with any one of SEQ ID NOs:40-44 and 46-56.

[0010] In some embodiments, guide RNA molecules targeting sequences contained in one of exons 3 or 8 of the HPRT1 gene have at least 95% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, guide RNA molecules targeting sequences contained in one of exons 3 or 8 of the HPRT1 gene have at least 96% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, guide RNA molecules targeting sequences contained in one of exons 3 or 8 of the HPRT1 gene have at least 97% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, guide RNA molecules targeting sequences contained in one of exons 3 or 8 of the HPRT1 gene have at least 98% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, guide RNA molecules targeting sequences contained in one of exons 3 or 8 of the HPRT1 gene have at least 99% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, guide RNA molecules targeting sequences contained in one of exons 3 or 8 of the HPRT1 gene comprise any one of SEQ ID NOs:40-44 and 46-56.

[0011] In some embodiments the endonuclease comprises a Cas protein. In some embodiments the Cas protein comprises a Cas9 protein. In some embodiments, the Cas protein comprises Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0012] In some embodiments, lymphocytes obtained from a donor sample are transfected or transduced by viral delivery vehicles, non-viral delivery vehicles, and / or physical methods. In some embodiments, the physical method is selected from microinjection and electroporation.

[0013] In some embodiments, the non-viral delivery vehicle is a nanocapsule. In some embodiments, nanocapsules optionally comprise at least one targeting moiety. In some embodiments, nanocapsules comprise at least one targeting moiety. In some embodiments, at least one targeting moiety targets any one of the human mesenchymal stem cell CD markers, including the CD29, CD44, CD90, CD49a-f, CD51, CD73(SH3), CD105(SH2), CD106, CD166, and Stro-1 markers. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127, FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0014] In some embodiments, costimulation by one or more costimulatory moieties may be used to activate target cells, including T cells. In some embodiments, co-stimulation can be achieved by activating one or more cell surface markers including, but not limited to, CD28, ICOS, CTLA4, PD1, PD1H, and BTLA. In some embodiments, the co-stimulatory moiety is an antibody.

[0015] In some embodiments, the viral delivery vehicle is an expression vector, wherein the expression vector comprises a first nucleic acid sequence encoding an endonuclease and a second nucleic acid sequence encoding a guide RNA molecule. In some embodiments, the expression vector is a lentiviral expression vector.

[0016] In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 70% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 75% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 80% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 85% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 90% compared to non-transfected donor lymphocytes. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 95% compared to non-transfected donor lymphocytes.

[0017] In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with a purine analogue. In some embodiments, the Purine Analogue is 6-TG. In some embodiments, the purine analogue is 6-mercaptopurine (6-MP). In some embodiments, the amount of Purine Analog ranges between about 1 and about 15 μg / mL. In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with both a purine analog (eg, in an amount ranging between about 1 and about 15 μg / mL) and allopurinol.

[0018] In some embodiments, at least about 70% of the engineered lymphocyte population is sensitive to a dihydrofolate reductase inhibitor. In some embodiments, at least about 80% of the engineered lymphocyte population is sensitive to a dihydrofolate reductase inhibitor. In some embodiments, the method further comprises administering to the patient one or more doses (eg, 2 or more doses, 3 or more doses, 4 or more doses, etc.) of a dihydrofolate reductase inhibitor. In some embodiments, the dihydrofolate reductase inhibitor is selected from the group consisting of MTX or MPA.

[0019] In some embodiments, the engineered lymphocyte population is administered as a single bolus. In some embodiments, multiple doses of the engineered lymphocyte population are administered to the patient (eg, 2 or more doses, 3 or more doses, 4 or more doses, etc.). In some embodiments, each dose of multiple doses is about 0.1 x 10 6 cells / kg ~ approx. 240 x 10 6 Contains cells / kg. In some embodiments, the total dose is about 0.1 x 10 6 cells / kg ~ approx. 730×10 6 Contains cells / kg.

[0020] A second aspect of the present disclosure is a method of providing the benefits of lymphocyte transfusion to a patient in need of such treatment while mitigating side reactions, wherein: (a) lymphocytes obtained from a donor sample are injected with (i) an endonuclease, and (ii) between about 134475181 and about 134475364 (based on genome build GRCh38, or equivalent positions in genome builds other than GRCh38) or between about 134498608 and about 134 generating a population of lymphocytes substantially deficient in HPRT by transfecting or transducing a guide RNA molecule targeting a sequence in the X chromosome located between 498684 (based on genome build GRCh38 or an equivalent position in genome builds other than GRCh38); (b) positively selecting the population of lymphocytes substantially deficient in HPRT ex vivo to provide a modified population of lymphocytes; There are methods comprising the step of administering to In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments, the method further comprises administering the HSC graft to the patient. In some embodiments, the HSC graft is administered prior to administration of the modified lymphocyte population, concurrently with administration of the modified lymphocyte population, or after administration of the modified lymphocyte population.

[0021] In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134475181 and about 134475364 based on the genome build GRCh38, or the equivalent position in genome builds other than GRCh38. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134475181 and about 134475364 based on genome build GRCh38, or at an equivalent position in genome builds other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0022] In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0023] In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 91% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 92% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 93% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 94% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule gene has at least 95% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 96% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule comprises any one of SEQ ID NOs:40-44 and 46-56.

[0024] In some embodiments the endonuclease comprises a Cas protein. In some embodiments the Cas protein comprises a Cas9 protein. In some embodiments, the Cas protein comprises Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0025] In some embodiments, lymphocytes obtained from a donor sample are transfected or transduced by viral delivery vehicles, non-viral delivery vehicles, and / or physical methods. In some embodiments, the physical method is selected from microinjection and electroporation.

[0026] In some embodiments, the non-viral delivery vehicle is a nanocapsule. In some embodiments, nanocapsules comprise at least one targeting moiety. In some embodiments, at least one targeting moiety targets any one of the human mesenchymal stem cell CD markers, including the CD29, CD44, CD90, CD49a-f, CD51, CD73(SH3), CD105(SH2), CD106, CD166, and Stro-1 markers. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127, FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0027] In some embodiments, costimulation by one or more costimulatory moieties may be used to activate target cells, including T cells. In some embodiments, costimulation can be achieved by activating one or more cell surface markers including, but not limited to, CD28, ICOS, CTLA4, PD1, PD1H, and BTLA. In some embodiments, the co-stimulatory moiety is an antibody.

[0028] In some embodiments, the viral delivery vehicle is an expression vector, wherein the expression vector comprises a first nucleic acid sequence encoding an endonuclease and a second nucleic acid sequence encoding a guide RNA molecule. In some embodiments, the expression vector is a lentiviral expression vector.

[0029] In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 70% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 75% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 80% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 85% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 90% compared to non-transfected donor lymphocytes.

[0030] In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with a purine analogue. In some embodiments, the Purine Analogue is 6-TG. In some embodiments, the Purine Analogue is 6-MP. In some embodiments, the amount of Purine Analog ranges between about 1 and about 15 μg / mL. In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with both a purine analogue and allopurinol.

[0031] In some embodiments, at least about 70% of the modified lymphocytes are sensitive to a dihydrofolate reductase inhibitor. In some embodiments, the method further comprises administering to the patient one or more doses of a dihydrofolate reductase inhibitor. In some embodiments, the dihydrofolate reductase inhibitor is selected from the group consisting of MTX or MPA.

[0032] In some embodiments, the modified lymphocytes are administered as a single bolus. In some embodiments, multiple doses of engineered lymphocytes are administered to the patient. In some embodiments, each dose of multiple doses is about 0.1 x 10 6 cells / kg ~ approx. 240 x 10 6 Contains cells / kg. In some embodiments, the total dose is about 0.1 x 10 6 cells / kg ~ approx. 730×10 6 Contains cells / kg.

[0033] In a third aspect of the present disclosure, a method of treating a hematologic cancer in a patient in need thereof comprises: (a) transfecting or transducing lymphocytes obtained from a donor sample with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in one of exons 3 or 8 of the HPRT1 gene to generate a population of lymphocytes substantially deficient in HPRT; (b) generating a population of lymphocytes substantially deficient in HPRT ex vivo. (c) inducing at least a partial graft-versus-malignancy effect by administering the HSC graft to the patient; and (d) administering a therapeutically effective amount of the modified lymphocyte population to the patient after detection of residual disease or disease recurrence. In some embodiments, the lymphocytes are T cells, preferably primary human T cells.

[0034] In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0035] In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0036] In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 91% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 92% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 93% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 94% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule gene has at least 95% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 96% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule comprises any one of SEQ ID NOs:40-44 and 46-56.

[0037] In some embodiments the Cas protein comprises a Cas9 protein. In some embodiments, the Cas protein comprises Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0038] In some embodiments, lymphocytes obtained from a donor sample are transfected or transduced by viral delivery vehicles, non-viral delivery vehicles, or physical methods. In some embodiments, the physical method is selected from microinjection and electroporation.

[0039] In some embodiments, the non-viral delivery vehicle is a nanocapsule. In some embodiments, nanocapsules comprise at least one targeting moiety. In some embodiments, at least one targeting moiety targets any one of the human mesenchymal stem cell CD markers, including the CD29, CD44, CD90, CD49a-f, CD51, CD73(SH3), CD105(SH2), CD106, CD166, and Stro-1 markers. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127, FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0040] In some embodiments, costimulation by one or more costimulatory moieties may be used to activate target cells, including T cells. In some embodiments, co-stimulation can be achieved by activating one or more cell surface markers including, but not limited to, CD28, ICOS, CTLA4, PD1, PD1H, and BTLA. In some embodiments, the co-stimulatory moiety is an antibody.

[0041] In some embodiments, the viral delivery vehicle is an expression vector, wherein the expression vector comprises a first nucleic acid sequence encoding an endonuclease and a second nucleic acid sequence encoding a guide RNA molecule. In some embodiments, the expression vector is a lentiviral expression vector.

[0042] In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 70% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 80% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 90% compared to non-transfected donor lymphocytes.

[0043] In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with a purine analogue. In some embodiments, the Purine Analogue is 6-TG. In some embodiments, the Purine Analogue is 6-MP. In some embodiments, the amount of Purine Analog ranges between about 1 and about 15 μg / mL. In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with both a purine analogue and allopurinol.

[0044] In some embodiments, at least about 70% of the modified lymphocytes are sensitive to a dihydrofolate reductase inhibitor. In some embodiments, the method further comprises administering to the patient one or more doses of a dihydrofolate reductase inhibitor. In some embodiments, the dihydrofolate reductase inhibitor is selected from the group consisting of MTX or MPA.

[0045] In some embodiments, the modified lymphocytes are administered as a single bolus. In some embodiments, multiple doses of engineered lymphocytes are administered to the patient. In some embodiments, each dose of multiple doses is about 0.1 x 10 6 cells / kg ~ approx. 240 x 10 6 Contains cells / kg. In some embodiments, the total dose is about 0.1 x 10 6 cells / kg ~ approx. 730×10 6 Contains cells / kg.

[0046] In a fourth aspect of the present disclosure, a method of treating a patient with HPRT-deficient lymphocytes comprises: (a) isolating the lymphocytes from a donor subject; (b) contacting the isolated lymphocytes with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in one of exons 3 or 8 of the HPRT1 gene; (d) administering a therapeutically effective amount of the modified lymphocyte preparation to the patient after hematopoietic stem cell transplantation; and (e) optionally administering a dihydrofolate reductase inhibitor after the development of graft-versus-host disease (GvHD) in the patient. In some embodiments, the lymphocytes are T cells, preferably primary human T cells.

[0047] In some embodiments, the dihydrofolate reductase inhibitor is selected from the group consisting of MTX or MPA. In some embodiments, the agent that positively selects HPRT-deficient lymphocytes comprises a purine analogue. In some embodiments, the Purine Analogue is 6-TG. In some embodiments, the amount of 6-TG ranges between about 1 and about 15 μg / mL.

[0048] In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0049] In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0050] In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 91% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 92% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 93% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 94% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule gene has at least 95% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 96% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule comprises any one of SEQ ID NOs:40-44 and 46-56.

[0051] In some embodiments the Cas protein comprises a Cas9 protein. In some embodiments, the Cas protein comprises Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0052] In some embodiments, lymphocytes obtained from a donor sample are transfected or transduced by viral delivery vehicles, non-viral delivery vehicles, or physical methods. In some embodiments, the physical method is selected from microinjection and electroporation.

[0053] In some embodiments, the non-viral delivery vehicle is a nanocapsule. In some embodiments, nanocapsules comprise at least one targeting moiety. In some embodiments, at least one targeting moiety targets any one of the human mesenchymal stem cell CD markers, including the CD29, CD44, CD90, CD49a-f, CD51, CD73(SH3), CD105(SH2), CD106, CD166, and Stro-1 markers. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127, FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0054] In some embodiments, costimulation by one or more costimulatory moieties may be used to activate target cells, including T cells. In some embodiments, co-stimulation can be achieved by activating one or more cell surface markers including, but not limited to, CD28, ICOS, CTLA4, PD1, PD1H, and BTLA. In some embodiments, the co-stimulatory moiety is an antibody.

[0055] In some embodiments, the viral delivery vehicle is an expression vector, wherein the expression vector comprises a first nucleic acid sequence encoding an endonuclease and a second nucleic acid sequence encoding a guide RNA molecule. In some embodiments, the expression vector is a lentiviral expression vector.

[0056] In some embodiments, the preparation is administered as a single bolus. In some embodiments, multiple dose preparations are administered to the patient. In some embodiments, each dose of the preparation is about 0.1 x 10 6 cells / kg ~ approx. 240 x 10 6 Contains cells / kg. In some embodiments, the total dosage of the preparation is about 0.1 x 10 6 cells / kg ~ approx. 730×10 6 Contains cells / kg.

[0057] A fifth aspect of the present disclosure is the use of a preparation comprising modified lymphocytes to provide the benefits of lymphocyte transfusion to a subject in need of such treatment, wherein the preparation comprising: (a) isolating lymphocytes from a donor subject; and (c) exposing the population of HPRT-deficient lymphocytes to an agent that positively selects for HPRT-deficient lymphocytes and providing a preparation of modified lymphocytes. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments, the subject is in need of treatment after hematopoietic stem cell transplantation.

[0058] In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0059] In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134498608 and about 134498684 based on the genome build GRCh38, or the equivalent position in genome builds other than GRCh38. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0060] In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 91% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 92% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 93% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 94% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule gene has at least 95% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 96% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule comprises any one of SEQ ID NOs:40-44 and 46-56.

[0061] A sixth aspect of the present disclosure is a kit comprising: (i) a guide RNA molecule having at least 90% sequence identity with any one of SEQ ID NOS: 40-61; and (ii) a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein. In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 91% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 92% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 93% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 94% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 95% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 96% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA molecule comprises any one of SEQ ID NOs:40-61.

[0062] A seventh aspect of the present disclosure is a kit comprising: (i) a guide RNA molecule targeting a sequence within the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38, and (ii) a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 90% complementary to a sequence in the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 95% complementary to a sequence in the X chromosome located between about 134475181 and about 134475364 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0063] An eighth aspect of the present disclosure is a kit comprising: (i) a guide RNA molecule targeting a sequence within the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38, and (ii) a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 90% complementary to a sequence in the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 95% complementary to a sequence in the X chromosome located between about 134498608 and about 134498684 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0064] A ninth aspect of the present disclosure is a nanocapsule comprising (i) a guide RNA molecule having at least 90% sequence identity with any one of SEQ ID NOS: 40-61; and (ii) a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0065] In some embodiments, the guide RNA has at least 91% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 92% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 93% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 94% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 96% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 97% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 98% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 99% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has the sequence of any one of SEQ ID NOs:40-61.

[0066] In some embodiments, nanocapsules comprise at least one targeting moiety. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127 or FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0067] In some embodiments, nanocapsules comprise a polymer shell. In some embodiments, polymeric nanocapsules comprise two different positively charged monomers, at least one neutral monomer, and a crosslinker.

[0068] A tenth aspect of the present disclosure is a host cell transfected with (i) a guide RNA molecule having at least 90% sequence identity with any one of SEQ ID NOS: 40-61; and (ii) a nanocapsule comprising a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0069] In some embodiments, the guide RNA has at least 91% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 92% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 93% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 94% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 96% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 97% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 98% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 99% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has any one of SEQ ID NOs:40-61. In some embodiments, nanocapsules comprise at least one targeting moiety. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127, FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0070] In some embodiments, nanocapsules comprise a polymer shell. In some embodiments, polymeric nanocapsules comprise two different positively charged monomers, at least one neutral monomer, and a crosslinker. In some embodiments, the host cells are primary T lymphocytes. In some embodiments, host cells are CEM cells.

[0071] An eleventh aspect of the present disclosure is the use of a modified lymphocyte preparation to provide the benefits of lymphocyte transfusion to a subject in need of such treatment after hematopoietic stem cell transplantation, wherein the modified lymphocyte preparation comprises: (a) isolating lymphocytes from a donor subject; (b) contacting the isolated lymphocytes with (i) a guide RNA molecule having at least 90% sequence identity to any one of SEQ ID NOS: 40-61; and (ii) nanocapsules containing Cas protein; and (c) generated by exposing a population of HPRT-deficient lymphocytes to an agent that positively selects HPRT-deficient lymphocytes to provide a preparation of modified lymphocytes. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0072] In some embodiments, the guide RNA has at least 91% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 92% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 93% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 94% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 97% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 98% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 99% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has any one of SEQ ID NOs:40-61. In some embodiments, nanocapsules comprise at least one targeting moiety. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127, FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28. In some embodiments, the lymphocytes are T cells, preferably primary human T cells.

[0073] In some embodiments, nanocapsules comprise a polymer shell. In some embodiments, polymeric nanocapsules comprise two different positively charged monomers, at least one neutral monomer, and a crosslinker.

[0074] A twelfth aspect of the present disclosure is a kit comprising: (a) (i) a guide RNA molecule having at least 90% sequence identity with any one of SEQ ID NOS: 40-61; and (ii) a nanocapsule comprising a Cas protein; and (b) a dihydrofolate reductase inhibitor. In some embodiments, the dihydrofolate reductase inhibitor is MTX or MPA. In some embodiments, the guide RNA has at least 91% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 92% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 93% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 94% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 97% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 98% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 99% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has any one of SEQ ID NOs:40-61.

[0075] A thirteenth aspect of the present disclosure provides a method of providing the benefits of lymphocyte transfusion to a patient in need of such treatment while mitigating side effects, comprising: (a) transfecting or transducing lymphocytes obtained from a donor sample with (i) an endonuclease and (ii) a guide RNA having at least 90% sequence identity to any one of SEQ ID NOS: 40-61 to generate a population of lymphocytes substantially deficient in HPRT; (b) substantially deficient in HPRT. positively selecting a population of lymphocytes ex vivo to provide an engineered population of lymphocytes; and (c) after administration of the HSC graft, administering to the patient a therapeutically effective amount of the engineered population of lymphocytes. In some embodiments, the guide RNA has at least 91% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 92% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 93% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 94% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 97% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 99% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA comprises any one of SEQ ID NOs:40-61. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments, the method further comprises administering the HSC graft to the patient. In some embodiments, the HSC graft is administered prior to administration of the modified lymphocyte population, concurrently with administration of the modified lymphocyte population, or after administration of the modified lymphocyte population.

[0076] A fourteenth aspect of the present disclosure is a method of providing the benefits of lymphocyte transfusion to a patient in need of such treatment while mitigating side effects, comprising: (a) transfecting or transducing lymphocytes obtained from a donor sample with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in one of exons 2, 3 or 8 of the HPRT1 gene to generate a population of lymphocytes substantially deficient in HPRT; (c) administering to the patient a therapeutically effective amount of the modified lymphocyte population. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments, the method further comprises administering the HSC graft to the patient. In some embodiments, the HSC graft is administered prior to administration of the modified lymphocyte population, concurrently with administration of the modified lymphocyte population, or after administration of the modified lymphocyte population. In some embodiments, the guide RNA has at least 90% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:40-61.

[0077] In some embodiments, the guide RNA targets sequences in exon 2. In some embodiments, the guide RNA has at least 90% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA targets a sequence in exon 3. In some embodiments, the guide RNA has at least 90% sequence identity with any one of SEQ ID NOs:50-54. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:50-54. In some embodiments, the guide RNA targets a sequence in exon 8. In some embodiments, the guide RNA has at least 90% sequence identity with any one of SEQ ID NOs:55-56. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:55-56.

[0078] A fifteenth aspect of the present disclosure provides a method of providing the benefits of lymphocyte transfusion to a patient in need of such treatment while reducing side effects, comprising: (a) transfecting or transducing lymphocytes from a donor sample with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in exon 2 of the HPRT1 gene to generate a population of lymphocytes substantially deficient in HPRT; (b) generating a population of lymphocytes substantially deficient in HPRT. and (c) administering the HSC graft to the patient; and (d) administering to the patient a therapeutically effective amount of the modified lymphocyte population after administration of the HSC graft. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 90% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 91% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 92% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 93% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 94% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 97% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 98% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 99% sequence identity with any one of SEQ ID NOs:45 and 57-61.

[0079] In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:45. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:57. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:58. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:59. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:60. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:61. In some embodiments, the lymphocytes are T cells, preferably primary human T cells.

[0080] In some embodiments, lymphocytes obtained from a donor sample are transfected or transduced by viral delivery vehicles, non-viral delivery vehicles, and / or physical methods. In some embodiments, the physical method is selected from microinjection and electroporation.

[0081] In some embodiments, the non-viral delivery vehicle is a nanocapsule. In some embodiments, nanocapsules optionally comprise at least one targeting moiety. In some embodiments, nanocapsules comprise at least one targeting moiety. In some embodiments, at least one targeting moiety targets any one of the human mesenchymal stem cell CD markers, including the CD29, CD44, CD90, CD49a-f, CD51, CD73(SH3), CD105(SH2), CD106, CD166, and Stro-1 markers. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127, FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0082] In some embodiments, costimulation by one or more costimulatory moieties may be used to activate target cells, including T cells. In some embodiments, costimulation can be achieved by activating one or more cell surface markers including, but not limited to, CD28, ICOS, CTLA4, PD1, PD1H, and BTLA. In some embodiments, the co-stimulatory moiety is an antibody.

[0083] In some embodiments, the viral delivery vehicle is an expression vector, wherein the expression vector comprises a first nucleic acid sequence encoding an endonuclease and a second nucleic acid sequence encoding a guide RNA molecule. In some embodiments, the expression vector is a lentiviral expression vector.

[0084] In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 70% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 75% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 80% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 85% compared to non-transfected donor lymphocytes. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 90% compared to non-transfected donor lymphocytes. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is reduced by at least about 95% compared to non-transfected donor lymphocytes.

[0085] In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with a purine analogue. In some embodiments, the Purine Analogue is 6-TG. In some embodiments, the purine analogue is 6-mercaptopurine (6-MP). In some embodiments, the amount of Purine Analog ranges between about 1 and about 15 μg / mL. In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with both a purine analog (eg, in an amount ranging between about 1 and about 15 μg / mL) and allopurinol.

[0086] In some embodiments, at least about 70% of the engineered lymphocyte population is sensitive to a dihydrofolate reductase inhibitor. In some embodiments, at least about 80% of the engineered lymphocyte population is sensitive to a dihydrofolate reductase inhibitor. In some embodiments, the method further comprises administering to the patient one or more doses (eg, 2 or more doses, 3 or more doses, 4 or more doses, etc.) of a dihydrofolate reductase inhibitor. In some embodiments, the dihydrofolate reductase inhibitor is selected from the group consisting of MTX or MPA.

[0087] In some embodiments, the engineered lymphocyte population is administered as a single bolus. In some embodiments, multiple doses of the engineered lymphocyte population are administered to the patient (eg, 2 or more doses, 3 or more doses, 4 or more doses, etc.). In some embodiments, each dose of multiple doses is about 0.1 x 10 6 cells / kg ~ approx. 240 x 10 6 Contains cells / kg. In some embodiments, the total dose is about 0.1 x 10 6 cells / kg ~ approx. 730×10 6 Contains cells / kg.

[0088] In a sixteenth aspect of the present disclosure, a method of treating a hematological cancer in a patient in need thereof comprises: (a) generating a population of lymphocytes substantially deficient in HPRT by transfecting or transducing lymphocytes obtained from a donor sample with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in exon 2 of the HPRT1 gene; (b) positively selecting and modifying the population of lymphocytes substantially deficient in HPRT ex vivo. (c) inducing at least a partial graft-versus-malignancy effect by administering the HSC graft to the patient; and (d) administering to the patient a therapeutically effective amount of the modified lymphocyte population after detection of residual disease or disease recurrence. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments the endonuclease is the Cas9 protein. In some embodiments the endonuclease is the Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0089] In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 90% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 91% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 92% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 93% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 94% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 97% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 98% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 99% sequence identity with any one of SEQ ID NOs:45 and 57-61.

[0090] In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:45. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:57. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:58. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:59. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:60. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:61. In some embodiments, the lymphocytes are T cells, preferably primary human T cells.

[0091] In a seventeenth aspect of the present disclosure, a method of treating a patient with HPRT-deficient lymphocytes comprises: (a) isolating lymphocytes from a donor subject; (b) contacting the isolated lymphocytes with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in exon 2 of the HPRT1 gene; (d) administering to the patient a therapeutically effective amount of the modified lymphocyte preparation after hematopoietic stem cell transplantation; and (e) optionally administering a dihydrofolate reductase inhibitor after the development of graft-versus-host disease (GvHD) in the patient. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments the endonuclease is the Cas9 protein. In some embodiments the endonuclease is the Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0092] In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 90% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 91% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 92% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 93% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 94% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 97% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 98% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 99% sequence identity with any one of SEQ ID NOs:45 and 57-61.

[0093] In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:45. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:57. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:58. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:59. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:60. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:61. In some embodiments, the lymphocytes are T cells, preferably primary human T cells.

[0094] An eighteenth aspect of the present disclosure is the use of a modified lymphocyte preparation to provide the benefits of lymphocyte transfusion to a subject in need of such treatment after hematopoietic stem cell transplantation, wherein the modified lymphocyte preparation comprises: (a) isolating lymphocytes from a donor subject; and (c) exposing a population of HPRT-deficient lymphocytes to an agent that positively selects for HPRT-deficient lymphocytes and providing a preparation of modified lymphocytes. In some embodiments, the lymphocytes are T cells, preferably primary human T cells. In some embodiments the endonuclease is the Cas9 protein. In some embodiments the endonuclease is the Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0095] In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 90% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 91% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 92% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 93% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 94% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 97% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 98% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 99% sequence identity with any one of SEQ ID NOs:45 and 57-61.

[0096] In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:45. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:57. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:58. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:59. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:60. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises SEQ ID NO:61. In some embodiments, the lymphocytes are T cells, preferably primary human T cells.

[0097] A nineteenth aspect of the present disclosure is a method of providing the benefits of lymphocyte transfusion to a patient in need of such treatment while mitigating side effects, wherein: (a) lymphocytes obtained from a donor sample are transfused with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence within the X chromosome located between about 134473409 and about 134473460 based on genome build GRCh38, or an equivalent position in a genome build other than GRCh38. (b) positively selecting the population of lymphocytes substantially deficient in HPRT ex vivo to provide an engineered population of lymphocytes; and (c) administering a therapeutically effective amount of the population of engineered lymphocytes to the patient. In some embodiments, the method further comprises administering the HSC graft to the patient. In some embodiments, the HSC graft is administered prior to administration of the modified lymphocyte population, concurrently with administration of the modified lymphocyte population, or after administration of the modified lymphocyte population. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134473409 and about 134473460 based on genome build GRCh38, or at an equivalent position in genome builds other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 14 nucleotides to about 30 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides.

[0098] A twentieth aspect of the present disclosure is a kit comprising: (i) a guide RNA molecule targeting a sequence within the X chromosome located between about 134473409 and about 134473460 based on GRCh38, or an equivalent position in a genome build other than GRCh38, and (ii) a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein. In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134473409 and about 134473460 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 90% complementary to a sequence in the X chromosome located between about 134473409 and about 134473460 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the guide RNA molecule is at least about 95% complementary to a sequence in the X chromosome located between about 134473409 and about 134473460 based on GRCh38, or an equivalent position in a genome build other than GRCh38. In some embodiments, the targeted sequence has a length ranging from about 16 nucleotides to about 28 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 18 nucleotides to about 26 nucleotides. In some embodiments, the targeted sequence has a length ranging from about 21 nucleotides to about 25 nucleotides. In some embodiments, the targeted sequence has a length of about 21 nucleotides. In some embodiments, the targeted sequence has a length of about 22 nucleotides. In some embodiments, the targeted sequence has a length of about 23 nucleotides. In some embodiments, the targeted sequence has a length of about 24 nucleotides. In some embodiments, the targeted sequence has a length of about 25 nucleotides.

[0099] A twenty-first aspect of the present disclosure provides a method of providing the benefits of lymphocyte transfusion to a patient in need of such treatment while mitigating side reactions, comprising: (a) transfecting or transducing lymphocytes obtained from a donor sample with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in one of exons 2, 3 or 8 of the HPRT1 gene to generate a population of lymphocytes substantially deficient in HPRT; (c) administering to the patient a therapeutically effective amount of the modified lymphocyte population.

[0100] In some embodiments, the method further comprises administering the HSC graft to the patient. In some embodiments, the HSC graft is administered prior to administration of the modified lymphocyte population, concurrently with administration of the modified lymphocyte population, or after administration of the modified lymphocyte population.

[0101] In some embodiments, the guide RNA molecule targets a sequence contained in exon 2 of the HPRT1 gene. In some embodiments, the guide RNA molecule targets a sequence contained in exon 3 of the HPRT1 gene. In some embodiments, the guide RNA molecule targets a sequence contained in exon 8 of the HPRT1 gene.

[0102] In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 99% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises any one of SEQ ID NOs:45 and 57-61.

[0103] In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 3 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:41-44, 46 and 50-51. In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 3 of the HPRT1 gene has at least 99% sequence identity with any one of SEQ ID NOs:41-44, 46 and 50-51. In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 3 of the HPRT1 gene comprises any one of SEQ ID NOs:41-44, 46 and 50-51.

[0104] In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 8 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:47-49, 46, 55 and 56. In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 8 of the HPRT1 gene has at least 99% sequence identity with any one of SEQ ID NOs:47-49, 46, 55 and 56. In some embodiments, a guide RNA molecule targeting a sequence contained in one of exons 8 of the HPRT1 gene comprises any one of SEQ ID NOs:47-49, 46, 55 and 56.

[0105] In some embodiments, the guide RNA molecule is at least about 85% complementary to a sequence in the X chromosome located between about 134475181 and about 134475364 based on genome build GRCh38, or at an equivalent position in genome builds other than GRCh38. In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134475181 and about 134475364 based on the genome build GRCh38, or the equivalent position in genome builds other than GRCh38.

[0106] In some embodiments the endonuclease comprises a Cas protein. In some embodiments the Cas protein comprises a Cas9 protein. In some embodiments, the Cas protein comprises Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0107] In some embodiments, lymphocytes obtained from a donor sample are transfected or transduced by viral delivery vehicles, non-viral delivery vehicles, or physical methods. In some embodiments, the physical method is selected from microinjection and electroporation. In some embodiments, the non-viral delivery vehicle is a nanocapsule, optionally comprising at least one targeting moiety. In some embodiments, the viral delivery vehicle is an expression vector, wherein the expression vector comprises a first nucleic acid sequence encoding an endonuclease and a second nucleic acid sequence encoding a guide RNA molecule. In some embodiments, the expression vector is a lentiviral expression vector.

[0108] In some embodiments, the level of HPRT1 gene expression within a population of lymphocytes substantially deficient in HPRT is at least about 70% reduced, preferably at least about 80% reduced, more preferably at least about 90% reduced, as compared to non-transfected donor lymphocytes.

[0109] In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with a purine analogue, preferably the purine analogue is selected from the group consisting of 6-TG and 6-MP. In some embodiments, the amount of Purine Analog ranges between about 1 and about 15 μg / mL. In some embodiments, positive selection comprises contacting the generated population of substantially HPRT-deficient lymphocytes with both a purine analogue and allopurinol.

[0110] In some embodiments, the method further comprises administering to the patient one or more doses of a dihydrofolate reductase inhibitor, preferably the dihydrofolate reductase inhibitor is selected from the group consisting of MTX or MPA.

[0111] In some embodiments, the engineered lymphocyte population is administered as a single bolus or multiple doses. In some embodiments, each dose of multiple doses is about 0.1 x 10 6 cells / kg ~ approx. 240 x 10 6 Contains cells / kg. In some embodiments, the total dose is about 0.1 x 10 6 cells / kg ~ approx. 730×10 6 Contains cells / kg.

[0112] In a twenty-second aspect of the present disclosure, a method of treating a patient with HPRT-deficient lymphocytes comprises: (a) isolating lymphocytes from a donor subject; (b) contacting the isolated lymphocytes with (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence contained in one of exons 2, 3 or 8 of the HPRT1 gene; (d) administering a therapeutically effective amount of the modified lymphocyte preparation to the patient after hematopoietic stem cell transplantation; and (e) optionally administering a dihydrofolate reductase inhibitor after the development of graft-versus-host disease (GvHD) in the patient. In some embodiments, the dihydrofolate reductase inhibitor is selected from the group consisting of MTX or MPA.

[0113] A twenty-third aspect of the disclosure is the use of a preparation of modified lymphocytes to provide the benefits of lymphocyte transfusion to a subject in need of such treatment, wherein the preparation of modified lymphocytes comprises: (a) isolating lymphocytes from a donor subject; and (c) exposing the HPRT-deficient population of lymphocytes to an agent that positively selects for HPRT-deficient lymphocytes and providing a preparation of modified lymphocytes. In some embodiments, the subject is in need of treatment after hematopoietic stem cell transplantation. In some embodiments, the guide RNA molecule targets a sequence within the X chromosome located between about 134475181 and about 134475364 based on the genome build GRCh38, or the equivalent position in genome builds other than GRCh38. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule targeting exon 3 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:41-44, 46 and 50-51. In some embodiments, the guide RNA molecule targeting exon 3 of the HPRT1 gene comprises any one of SEQ ID NOs:41-44, 46 and 50-51. In some embodiments, the guide RNA molecule targeting exon 8 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs:47-49, 55 and 56. In some embodiments, the guide RNA molecule targeting exon 8 of the HPRT1 gene comprises any one of SEQ ID NOs:47-49, 55 and 56.

[0114] A twenty-fourth aspect of the present disclosure provides a method of providing the benefits of lymphocyte transfusion to a patient in need of such treatment while reducing side effects, comprising: (a) transfecting or transducing lymphocytes obtained from a donor sample with (i) an endonuclease, and (ii) a guide RNA having at least 90% sequence identity to any one of SEQ ID NOs: 40-61 to generate a population of lymphocytes substantially deficient in HPRT; (b) substantially deficient in HPRT. and (c) administering a therapeutically effective amount of the modified lymphocyte population to the patient. In some embodiments, the method further comprises administering the HSC graft to the patient. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA comprises any one of SEQ ID NOs:40-61.

[0115] A twenty-fifth aspect of the present disclosure is a kit comprising: (i) a guide RNA molecule having at least 95% sequence identity with any one of SEQ ID NOS: 40-61; and (ii) a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein.

[0116] A twenty-sixth aspect of the present disclosure is a kit comprising: (i) a guide RNA molecule targeting a sequence in the X chromosome located between about 134475181 and about 134475364 based on genome build GRCh38, or an equivalent position in a genome build other than GRCh38, and (ii) a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9 protein and Cas12 protein.

[0117] In contrast to other "off-switch" methods, hematopoietic cells (including T cells) treated according to the disclosed methods do not need to express a "suicide gene." Rather, the methods of the present disclosure provide knockdown or knockout of endogenous genes that do not cause undesirable effects on blood cells and overall superior results. Applicants submit that according to the methods described herein, given ex vivo 6-TG chemoselection of a population of genetically modified cells, HSCs (or lymphocytes), is provided for administration to a subject that allows quantitative depletion of the cells in vivo by administering a dihydrofolate reductase inhibitor (e.g., methotrexate (MTX)). In addition, treatments described in the methods of the present disclosure provide potentially higher doses of donor T cells and more aggressive therapy than therapies that do not incorporate a "kill switch." Furthermore, the use of dihydrofolate reductase inhibitors to modulate the number of engineered T cells is clinically compatible with existing methods of treating GvHD, i.e., MTX is used to help alleviate GvHD syndrome in patients who do not receive the engineered T cells of the present disclosure.

[0118] Applicants believe that compared to donor lymphocytes transduced with the herpes simplex thymidine kinase gene, the treatment described in the methods of the present disclosure results in cell ablation and alleviates limitations, including immunogenicity, which precludes the possibility of future transfusions (Zhou X, Brenner MK, “Improving the safety of T-Cell therapies using an inducible caspase-9 gene,” Exp Hematol. 2016 Nov;44(11):101). 3-1019, the disclosure of which is incorporated herein by reference in its entirety). Applicants also propose that this method allows the use of ganciclovir for concurrent clinical conditions other than GvHD that do not result in unwanted clearance of HSV-tk donor lymphocytes (e.g., ganciclovir is not excluded from administering control CMV infections that are common in the allo-HSCT setting when the recently described method is utilized). [Brief description of the drawing]

[0119]

Figure 1

Figure 2

[0120] array list Nucleic acid and amino acid sequences attached herein are presented using standard letter abbreviations for nucleotide bases and the three-letter code for amino acids, as defined in 37 C.F.R. 1.822. The Sequence Listing has been submitted as a 30KB ASCII text file named "Calimmune-097WO_ST25.txt" created June 25, 2021, which is incorporated herein by reference. [Mode for carrying out the invention]

[0121] definition It is also to be understood that for any method claimed herein involving more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited, unless expressly indicated to the contrary.

[0122] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0123] As used herein in the specification and claims, with reference to a list of one or more elements, the phrase "at least one" means at least one element selected from any one or more elements of the list of elements, but specifically does not necessarily include at least one of each and every element recited in the list of elements, nor exclude any combination of the elements of the list of elements. This definition also permits that elements may optionally represent other elements than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not they are related to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B", or equivalently "at least one of A and / or B") includes, in one embodiment, at least one, and optionally more than one, A in which B is absent (and optionally includes elements other than B); yet another embodiment includes at least one and optionally more than one A, and at least one and optionally more than one B (and optionally other elements);

[0124] As used herein, the terms "comprising," "including," "having," etc. are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," etc. are used interchangeably and have the same meaning. In particular, each term is to be interpreted as an open term meaning "at least no more than" and is also to be interpreted as not excluding additional features, limitations, aspects, and the like. Thus, for example, "a device having components a, b, and c" means a device that includes at least components a, b, and c. Similarly, the phrase: "a method comprising steps a, b, and c" means that the method comprises at least steps a, b, and c. Additionally, although steps and methods are outlined herein in a particular order, those skilled in the art will recognize that the order of steps and methods may vary.

[0125] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" provided above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, i.e., including at least one inclusive, but including some element or more than one of the list of elements, and possibly also items not in the further list. Only the expressly specified terms to the contrary, such as "only one of" or "exactly one of," or when used in the claims, "consisting of" refer to the inclusion of exactly one element of some element or list of elements. In general, as used herein, the term “or”, when preceded by an exclusive term such as “either,” “one of,” “only one of,” or “exactly one of,” should only be construed to indicate exclusive alternatives (i.e., “one or the other but not both”). When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0126] As used herein, the term "administer" or "administering" means providing a composition, formulation, or particular agent, including those described herein, to a subject (e.g., a human patient) in need of treatment.

[0127] As used herein, the term "Cas protein" refers to an RNA-guided nuclease comprising a Cas protein, or fragment thereof. Cas proteins are also called CRISPR (clustered regularly interspaced short palindromic repeat)-related nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). A CRISPR cluster contains a spacer, sequence complementarity to the preceding mobile element, and a target invasion nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Cas proteins include, but are not limited to, Cas9 proteins, Cas9-like proteins encoded by Cas9 orthologs, Cas9-like synthetic proteins, Cpf1 proteins, proteins encoded by Cpf1 orthologs, Cpf1-like synthetic proteins, C2c1 proteins, C2c2 proteins, C2c3 proteins, and variants and modifications thereof. Further examples of Cas proteins include, but are not limited to, Cpf1, C2c1, C2c3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, and Casl3c. Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm 6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas 12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c.

[0128] In some embodiments, the Cas protein is a Class 2 CRISPR-related protein. As defined herein, a "class 2 type CRISPR-Cas system" refers to a CRISPR-Cas system that functions with a single protein as an effector complex (eg Cas9). As defined herein, a "Class 2 Type II CRISPR-Cas system" refers to a CRISPR-Cas system that includes the Cas9 gene among its cas genes. "Class 2 type II-A CRISPR-Cas system" refers to a CRISPR-Cas system containing the cas9 and Csn2 genes. "Class 2 type II-B CRISPR-Cas system" refers to a CRISPR-Cas system containing the cas9 and cas4 genes. A "class 2 type II-C CRISPR-Cas system" refers to a CRISPR-Cas system that contains the Cas9 gene but neither the Csn2 nor the Cas4 gene. A "Class 2 Type V CRISPR-Cas system" refers to a CRISPR-Cas system that contains the cas12 gene (Cas12a, 12b or 12c gene) in its cas gene. A "Class 2 Type VI CRISPR-Cas system" refers to a CRISPR-Cas system that includes the Cas13 gene (Cas13a, 13b or 13c gene) in its Cas gene. Each wild-type Cas protein interacts with one or more cognate polynucleotides (most typically RNA) to form a nucleoprotein complex (most typically a ribonucleoprotein complex). Additional Cas proteins are described by Haft et al., A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR / Cas Subtypes Exist in Prokaryotic Genomes, PLoS Comput. Biol., 2005, Nov;1(6):e60. is an item.

[0129] As used herein, the term “Cas9” or “Cas9 protein” refers to an enzyme (wild-type or recombinant) that can exhibit modest endonuclease activity (e.g., cleaving phosphodiester bonds within polynucleotides) guided by CRSPR RNA (crRNA) having a sequence complementary to a target polynucleotide. Cas9 polypeptides are known in the art and include, for example, Cas9 polypeptides from any of a variety of biological origins, including prokaryotic origins such as bacteria and archaea. Bacterial Cas9 includes Actinomycota (e.g., Actinomyces nassurundi) Cas9, Akwifex Cas9, Bacteroidetes Cas9, Chlamydia Cas9, Green Nonsulphur Bacteria Cas9, Cyanobacteria Cas9, Ercimicrobium Cas9, Fibrobacter Cas9, Firmiculus Cas9 (e.g. Ptococcus thermophilus Cas9, Listeria innocua Cas9, Streptococcus agalactia Cas9, Streptococcus mutans Cas9, and Enterococcus faecium Cas9), Fusobacterium phylum Cas9, Proteobacteria (e.g. meningococcus, Campylobacter jejuni and Campylobacter rarii) Cas9, spirochetes (e.g. Neema denticola) including Cas9, etc. Archaeal Cas9 includes Lilium Archaea Cas9 (eg, Methanococcus maripaldis Cas9) and the like. Various Cas9 and related polypeptides are known, e.g., Makarova et al. (2011) Nature Reviews Microbiology 9:467-477, Makarova et al. (2011) Biology Direct 6:38, Haft et al. (2005) PLOS Computational Biology I: e60 and Chylinski et al. (2015) Nat.Rev.Microbio.13:722-736; and B. Zetsche et al., Cpf1 is a single RNA-guided endonuclease of a class2 CRISPR-Cas system. (2015) Cell.163(3):759-771.

[0130] Other Cas9 polypeptides include Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Mycoplasma gallisepticum F strain Cas9, Nitratifructor sarsuginis DSM16511 strain Cas9, Parvivacrum lavamentivorans Cas9, Rosebria intestinalis Cas9, Neisseria cinerea Cas9. 9, Gluconacetobacter diazotrophicus Cas9, Azospirillum sp. B510 Cas9, Sphaerochaeta globus badi strain cas9, Flavobacterium columnar Cas9, Flavicola tafensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus falciminis Cas9, Streptococcus Pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus schudinthermedius Cas9, Filifactor arrokis Cas9, Treponema denticola Cas9, Legionella pneumophila paris lineage Cas9, Stella wadsworthensis Cas9, and Corynebacter diphtheriae Cas9. The term "Cas9" includes any Cas9 family Cas9 polypeptide, including any isoform of Cas9. Various Cas9 homologues, orthologs, and variant amino acid sequences beyond those specifically described or provided herein are known, commonly available, and within the scope of those skilled in the art, and thus within the spirit and scope of the present disclosure.

[0131] As used herein, the term "Cas12" or "Cas12 protein" refers to any Cas12 protein, including but not limited to Cas12 proteins such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e. In some embodiments, the Cas12 protein is a functional Cas12 protein, in particular Cas12a / Cpf1 protein from Acidaminococcus strain BV3L6 (Uniprot Entry: U2UMQ6; Uniprot Entry Name: CS12A_ACISB) or Cas12a / Cpf1 protein from Francisella tularensis (Uniprot Entry: A0Q7Q2; Uniprot Entry Name: CS12A_FRATN). It has an amino acid sequence that is at least 85% (or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the amino acid sequence of the 12a / Cpf1 protein. In some embodiments, the Cas12 protein is a Cas12 polypeptide that is substantially identical to a protein found in nature, or a Cas12 polypeptide that has at least 85% sequence identity (or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity) with a Cas12 protein found in nature and has substantially the same biological activity. get. Examples of Cas12a proteins include, but are not limited to, FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; Cas12a is preferably LbCas12a. Examples of Cas12b proteins include, but are not limited to, AacCas12b, Aac2Cas12b, AkCas12b, AmCas12b, AhCas12b, AcCas12b.

[0132] As used herein, the phrase "effective amount" refers to that amount of a composition or formulation described herein that will elicit a diagnostic, biological or medical response in a tissue, system, animal, or human as contemplated by a researcher, veterinarian, physician, or other medical practitioner.

[0133] As used herein, the term "electroporation" refers to a technique that applies an electric field to cells to increase the permeability of cell membranes, allowing the introduction of chemicals, small molecules, proteins, nucleic acids, etc. into cells.

[0134] As used herein, the term "expression cassette" refers to one or more gene sequences within a vector that can subsequently express RNA and, in some embodiments, protein. An expression cassette contains at least one promoter and at least one gene of interest. In some embodiments, an expression cassette comprises at least one promoter, at least one gene of interest, and at least one additional nucleic acid sequence that encodes a molecule for expression (eg, RNAi). In some embodiments, the expression cassette is oriented within the vector such that the nucleic acid within the cassette is transcribed into RNA, translated into a protein or polypeptide as required, undergoes the appropriate post-translational modifications necessary for activity in transformed cells (e.g., transduced stem cells), and is translocated into the appropriate intracellular compartment or secreted into the extracellular compartment to the appropriate compartment for biological activity. In some embodiments, the cassette has its 3' and 5' ends adapted to facilitate insertion into a vector, e.g., a restriction endonuclease site at each end.

[0135] As used herein, the term "functional nucleic acid" refers to a molecule that has the ability to reduce protein expression by directly interacting with a protein-encoding transcript. siRNA molecules, ribozymes, and antisense nucleic acids constitute exemplary functional nucleic acids.

[0136] As used herein, the term "gene" refers broadly to any segment of DNA associated with biological function. Genes include, but are not limited to, sequences comprising coding sequences, promoter regions, cis-regulatory sequences, non-expressed DNA segments that are specific recognition sequences for regulatory proteins, non-expressed DNA segments that contribute to gene expression, DNA segments designed to have desired parameters, or combinations thereof.

[0137] As used herein, the term "gene silencing" is meant to describe down-regulation, knockdown, degradation, inhibition, suppression, suppression, prevention, or reduction in expression of a gene, transcript and / or polypeptide product. Gene silencing and interference also describes the prevention of translation of mRNA transcripts into polypeptides. In some embodiments, translation is prevented, inhibited, or reduced by degradation of mRNA transcripts or blocking of mRNA translation.

[0138] As used herein, the term "gene expression" refers to the cellular process by which a biologically active polypeptide is produced from a DNA sequence.

[0139] As used herein, the term "genome build" refers to successive "versions" of the human genome reference. The latest build of the Human Reference Genome has been named GRCh38 (for Genome Research Consortium human build 38), but is commonly referred to by the colloquial name Hg38 (for Human genome build 38).

[0140] As used herein, the term "guide RNA" or "gRNA" refers to an RNA molecule capable of targeting a CRISPR effector with nuclease activity to cleave a specific target nucleic acid.

[0141] As used herein, the term "hematopoietic cell transplant" or "hematopoietic cell transplantation" refers to bone marrow transplantation, peripheral blood stem cell transplantation, umbilical vein blood transplantation, or any other source of multipotent hematopoietic stem cells. Similarly, the term "stem cell transplant" or "transplant" refers to a composition comprising stem cells in contact (eg, suspended) with a pharmaceutically acceptable carrier. Such compositions can be administered to a subject through a catheter.

[0142] As used herein, the term "host cell" refers to a cell that is modified using the methods of the present disclosure. In some embodiments, the host cell is a mammalian cell in which the expression vector is expressed. Suitable mammalian host cells include, but are not limited to, human cells, mouse cells, non-human primate cells (e.g., rhesus monkey cells), human progenitor or stem cells, 293 cells, HeLa cells, D17 cells, MDCK cells, BHK cells, and Cf2Th cells. In certain embodiments, host cells comprising expression vectors of the present disclosure are hematopoietic cells such as hematopoietic progenitor / stem cells (e.g., CD34-positive hematopoietic progenitor / stem cells), monocytes, macrophages, peripheral blood mononuclear cells, CD4+ T lymphocytes, CD8+ T lymphocytes, or dendritic cells. Hematopoietic cells (eg, CD4+ T lymphocytes, CD8+ T lymphocytes, and / or monocytes / macrophages) transduced by expression vectors of the present disclosure can be allogeneic, autologous, or from a matched sibling. The hematopoietic progenitor / stem cells, in some embodiments, are CD34 positive and isolated from the patient's bone marrow or peripheral blood. Isolated CD34-positive hematopoietic progenitor / stem cells (and / or other hematopoietic cells described herein) are, in some embodiments, transduced with an expression vector as described herein.

[0143] As used herein, the term "hypoxanthine-guanine phosphoribosyltransferase" or "HPRT" refers to an enzyme involved in purine metabolism encoded by the HPRT1 gene (see, eg, SEQ ID NO:12). HPRT1 is located on the X chromosome and is therefore present in single copy in males. HPRT1 encodes a transferase that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate by transferring the 5-phosphoroibosyl group from 5-phosphoribosyl-1-pyrophosphate to a purine. Enzymes primarily function to recycle purines from degraded DNA for use in new purine synthesis.

[0144] As used herein, the term "indel" refers to mutations named by a mixture of insertions and deletions. It refers to the length difference between two alleles when it is not known whether the difference was originally caused by a sequence insertion or by a sequence deletion. If the number of nucleotides inserted / deleted is not divisible by 3 and occurs in a protein-coding region, it is also a frameshift mutation (frameshift mutations generally lead to reading of codons following mutations that encode different amino acids).

[0145] As used herein, the term "lentivirus" refers to a genus of retroviruses capable of infecting dividing and non-dividing cells. Some examples of lentiviruses include HIV (human immunodeficiency virus: HIV type 1, and HIV type 2), the causative agent of human acquired immunodeficiency syndrome (AIDS); Visna maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, goat arthritic encephalitis virus, which causes immunodeficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia and encephalopathy in horses; equine infectious anemia virus, which causes immunodeficiency in cats , feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV), which can cause lymphadenopathy, lymphocytosis, and central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in great primates.

[0146] As used herein, the term "lentiviral vector" is used to denote any form of lentiviral-derived nucleic acid that is used to transfer genetic material into cells by transduction. The term encompasses lentiviral vector nucleic acids such as DNA and RNA, encapsulated forms of these nucleic acids, and viral particles in which the viral vector nucleic acids are packaged.

[0147] As used herein, the term "lymphocyte" refers to one or more subtypes of white blood cells in the vertebrate immune system, including T cells, B cells and natural killer (NK) cells. Those skilled in the art will appreciate that T cells (also called T lymphocytes or CD3+ T lymphocytes) can be characterized based on their specific functions: helper / effector (CD4 T cells), cytotoxic (CD8 T cells), memory, regulatory and gamma delta (γδ) T cells. Those skilled in the art will further appreciate that T cell types can be distinguished by the type and distribution of cell surface markers. By way of example, subpopulations of T cells can be distinguished by the cell surface markers CD4 and CD8 and CC chemokine receptor 7 (CCR7) and CD45RA. Such subpopulations are further distinguishable by the expression of other cell surface markers. For example, naive T cells, effector memory (EM), central memory (CM), and effector T cell subsets can be further defined by CCR7 and CD45RA expression in addition to other markers. In some embodiments, lymphocytes are T cells. In some embodiments, lymphocytes are B cells. In some embodiments, the lymphocytes are natural killers (NK). In some embodiments, the lymphocytes are primary human T cells. In some embodiments, lymphocytes are CD3+ T cells. In some embodiments, lymphocytes are CD4+ T cells. In some embodiments, lymphocytes are CD8+ T cells. In some embodiments, lymphocytes are HLA-DR+ T cells. In some embodiments, the lymphocytes are αβ T cells. In some embodiments, the lymphocytes are γδT cells.

[0148] As used herein, the term "knock down" or "knockdown" when used in reference to the effect of RNAi on gene expression means that the level of gene expression is inhibited and reduced below levels normally observed when examined under substantially the same conditions but in the absence of RNAi.

[0149] As used herein, the terms "knock-out" or "knockout" refer to partial or complete suppression of the expression of an endogenous gene. This is generally accomplished by deleting part of the gene or replacing it with a second sequence, but it can also be caused by other modifications to the gene such as the introduction of stop codons, mutation of key amino acids, removal of intron junctions, etc. Thus, a "knockout" construct is a nucleic acid sequence, such as a DNA construct, that, when introduced into a cell, results in suppression (partial or complete) of expression in the cell of a polypeptide or protein encoded by endogenous DNA. In some embodiments, a "knockout" includes mutations such as point mutations, insertions, deletions, frameshifts, or missense mutations.

[0150] As used herein, the term "microinjection" refers to techniques for introducing chemicals, small molecules, proteins, nucleic acids, etc. into single cells by micropipette insertion into the cells of interest.

[0151] As used herein, the term "multiplicity of infection" or "MOI" refers to the ratio of agent (eg, phage or more generally virus, bacteria) to infection target (eg, cell). For example, when referring to a population of cells inoculated with virus particles, the multiplicity of infection or MOI is the ratio of the number of virus particles to the number of target cells present in a given space.

[0152] As used herein, the term "minicell" refers to an anucleated form of bacterial cell resulting from impaired coordination of cell division with DNA segregation during binary fission. Minicells are not dependent on specific gene rearrangements or episomal gene expression, unlike other vesicles that are generated and released spontaneously under specific circumstances and in contrast to minicells. Minicells of the present disclosure are anucleate forms of E. coli or other bacterial cells that arise from impaired coordination of cell division with DNA segregation during binary fission. Prokaryotic chromosomal replication is associated with normal binary fission, including intermediate cell membrane formation. In E. coli, for example, mutation of the min gene, such as minCD, can remove the inhibition of septum formation at the cell poles during cell division, resulting in the production of normal daughter cells and nucleated minicells. See de Boer et al., 1992; Raskin & de Boer, 1999; Hu & Lutkenhaus, 1999; Harry, 2001. Minicells are not dependent on specific gene rearrangements or episomal gene expression, unlike other vesicles that are generated and released spontaneously under specific circumstances and in contrast to minicells. To practice the present disclosure, minicells desirably have intact cell walls (“intact minicells”). In addition to min operon mutations, anucleated minicells are also generated in a range of other gene rearrangements or mutations that affect septum formation, such as divIVB1 in Bacillus subtilis. See Reeve and Cornett, 1975; Levin et al., 1992. Minicells are also formed after perturbation of the level of gene expression of proteins involved in cell division / chromosomal segregation. For example, overexpression of minE results in polar division and production of minicells. Similarly, chromosome-less minicells can result from defects in chromosomal segregation, such as the smc mutation in B. subtilis (Britton et al., 1998), the spoOJ deletion in B. subtilis (Ireton et al., 1994), the mukB mutation in E. coli (Hiraga et al., 1989), and the parC mutation in E. coli (Stewart and D'Ari, 1992). The gene product is supplied in trans. When overexpressed from a high-copy number plasmid, for example, CafA can increase the rate of cell division and / or inhibit post-replicative chromosome segregation (Okada et al., 1994), leading to the formation of chained cells and anucleated minicells (Wachi et al., 1989; Okada et al., 1993). Minicells are prepared from any bacterial cell of Gram-positive or Gram-negative origin.

[0153] As used herein, the term "mutation" refers to a change in a sequence, such as a nucleotide or amino acid sequence, from a native, wild-type, standard or reference version of the respective sequence, ie, a non-mutated sequence. A mutated gene can result in a mutated gene product. A mutated gene product differs from a non-mutated gene product in one or more amino acid residues. In some embodiments, a mutated gene that results in a mutated gene product can have about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more sequence identity with the corresponding non-mutated nucleotide sequence.

[0154] As used herein, the term “nanocapsule” refers to a nanoparticle having a shell, such as a polymer shell, that encapsulates one or more components, such as one or more proteins and / or one or more nucleic acids. In some embodiments, the nanocapsules have an average diameter of about 200 nanometers (nm) or less, such as between about 1 and 200 nm, or between about 5 and about 200 nm, or between about 10 and about 150 nm, or between about 15 and 100 nm, or between about 15 and about 150 nm, or between about 20 and about 125 nm, or between about 50 and about 100 nm, or between about 50 and about 75 nm. In other embodiments, the nanocapsules are about 10 nm to about 20 nm, about 20 nm to about 25 nm, about 25 nm to about 30 nm, about 30 nm to about 35 nm, about 35 nm to about 40 nm, about 40 nm to about 45 nm, about 45 nm to about 50 nm, about 50 nm to about 55 nm, about 55 nm to about 60 nm, about 60 nm to about 65 nm, about 70 nm to about 75 nm, about 70 nm to about 75 nm. It has an average diameter of between 5 nm and about 80 nm, about 80 nm and about 85 nm, about 85 nm and about 90 nm, about 90 nm and about 95 nm, about 95 nm and about 100 nm, or about 100 nm and about 110 nm. In some embodiments, the nanocapsules are designed to degrade in about 1 hour, or about 2 hours, or about 3 hours, or about 4 hours, or about 5 hours, or about 6 hours, or about 12 hours, or about 1 day, or about 2 days, or about 1 week, or about 1 month. In some embodiments, the surface of the nanocapsules can have a charge (eg, measured in standard phosphoric acid solution) between about 1 and about 15 millivolts (mV). In other embodiments, the surface of the nanocapsules can have a charge between about 1 and about 10 mV.

[0155] As used herein, the term "positively charged monomer" or "cationic monomer" refers to a monomer having a net positive charge, ie +1, +2, +3. In some embodiments, a positively charged monomer is a monomer that includes a positively charged group. As used herein, the term "negatively charged monomer" or "anionic monomer" refers to a monomer with a net negative charge, ie, -1, -2, -3. In some embodiments, a negatively charged monomer is a monomer that includes a negatively charged group. As used herein, the term "neutral monomer" refers to a monomer that has a net neutral charge.

[0156] As used herein, the term "polymer" is defined to be inclusive of homopolymers, copolymers, interpenetrating polymer networks, and oligomers. Accordingly, the term polymer is used interchangeably herein with the terms homopolymer, copolymer, interpenetrating polymer network, and the like. The term "homopolymer" is defined as a polymer derived from a single species of monomer. The term "copolymer" defines a polymer derived from more than one species of monomers, including copolymers obtained by copolymerization of two monomer species, copolymers obtained from three monomer species ("terpolymers"), copolymers obtained from four monomer species ("quaterpolymers"), and the like. The term "copolymer" is further defined to be inclusive of random copolymers, alternating copolymers, graft copolymers, and block copolymers. Copolymers, as the term is commonly used, include interpenetrating polymer networks. The term "random copolymer" is defined as a copolymer containing macromolecules in which the likelihood of finding a given monomeric unit at any given site in the chain does not depend on the properties of adjacent units. In random copolymers, the sequence distribution of the monomer units follows Bernoulli statistics. The term "alternating copolymer" is defined as a copolymer comprising macromolecules containing two types of monomeric units in an alternating arrangement.

[0157] As used herein, the term "crosslinker" refers to a bond or moiety that provides a connection (e.g., intramolecular or intermolecular linkage) between two or more molecular chains, domains, or other moieties. In some embodiments, a cross-linker is a molecule that forms a link between molecular chains forming a linked molecule.

[0158] As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, enhancer or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and / or translation of the nucleic acid corresponding to the second sequence when an appropriate molecule (e.g., a transcriptional activator protein) binds to the expression control sequence.

[0159] As used herein, the term "promoter" refers to a polynucleotide (DNA or RNA) recognition site to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In some embodiments, promoters that operate in mammalian cells comprise an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated and / or another sequence found 70-80 bases upstream from the start of transcription, a CNCAAT region where N can be any nucleotide.

[0160] As used herein, the term "pharmaceutically acceptable carrier or excipient" generally refers to a safe, non-toxic, and non-biological or undesirable carrier or excipient useful in the preparation of pharmaceutical formulations, and includes carriers or excipients that are acceptable for veterinary use as well as human pharmaceutical use.

[0161] As used herein, the term "retrovirus" refers to a virus whose RNA genome is reverse transcribed by retroviral reverse transcriptase into a cDNA copy that is integrated into the host cell genome. Retroviral vectors and methods of making retroviral vectors are known in the art. Briefly, to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in place of specific viral sequences to produce a virus that is replication defective. To produce virions, a packaging cell line is constructed that contains the gag, pol, and env genes but lacks the LTR and packaging components (Mann et al., Cell, Vol. 33:153-159, 1983). When a recombinant plasmid containing the cDNA together with the retroviral LTR and packaging sequences is introduced into this cell line, the packaging sequences cause the RNA transcripts of the recombinant plasmid to be packaged into virus particles, which are then secreted into the culture medium. Medium containing the recombinant retrovirus is then harvested, optionally concentrated, and used for gene transfer.

[0162] As used herein, the term “siRNA” or “small interfering RNA” refers to a short double-stranded RNA consisting of about 10 to several tens of nucleotides, which induces RNAi (RNA interference), i.e., induces degradation of target mRNA or inhibits target gene expression by cleaving target mRNA. RNA interference (“RNAi”) is a method of post-transcriptional inhibition of gene expression conserved in many eukaryotes, and refers to a phenomenon in which double-stranded RNA composed of a sense RNA having a sequence homologous to the mRNA of a target gene and an antisense RNA having a sequence complementary thereto is introduced into a cell or the like, thereby selectively inducing degradation of the mRNA of the target gene or inhibiting expression of the target gene. RNAi is induced by short (ie, less than about 30 nucleotides) double-stranded RNA molecules present in cells (Fire A. et al., Nature, 391:806-811, 1998). When the siRNA is introduced into a cell, it will inhibit the expression of target gene mRNA having a nucleotide sequence complementary to that of the siRNA.

[0163] As used herein, the term "small hairpin RNA" or "shRNA" refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a double stem. After post-transcriptional processing, small hairpin RNAs are converted into small interfering RNAs by cleavage events mediated by enzymes that are members of the RNase III family. As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription and is mediated, for example, by enzymes and / or Drosha.

[0164] As used herein, the term "subject" refers to mammals such as humans, mice or primates. Typically the mammal is a human (Homo sapiens).

[0165] As used herein, the terms "substantially deficient in HPRT" and variations thereof refer to cells, eg, host cells, in which the level of HPRT1 gene expression is reduced by at least about 50%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 55%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 60%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 65%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 70%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 75%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 80%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 85%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 90%. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 95%. In other embodiments, the remaining HPRT1 gene expression is up to about 40%. In other embodiments, the remaining HPRT1 gene expression is up to about 35%. In other embodiments, the remaining HPRT1 gene expression is up to about 30%. In other embodiments, the remaining HPRT1 gene expression is up to about 25%. In other embodiments, the remaining HPRT1 gene expression is up to about 20%. In other embodiments, the remaining HPRT1 gene expression is up to about 15%. In other embodiments, the remaining HPRT1 gene expression is up to about 10%.

[0166] As used herein, the terms "transduce" or "transduction" refer to gene delivery using viral or retroviral vectors by means of infection rather than transfection. For example, an anti-HPRT1 gene carried by a retroviral vector (a modified retrovirus used as an expression vector for introduction of nucleic acid into cells) is transduced into cells by infection and proviral integration. Thus, a "transduced gene" is a gene that has been introduced into a cell by retroviral or vector infection and proviral integration. A viral vector (eg, a "transducing vector") transduces genes into a "target cell," or host cell.

[0167] As used herein, the term "transfection" refers to methods of introducing naked DNA into cells by non-viral methods.

[0168] As used herein, the term "transduction" refers to the introduction of foreign DNA into the genome of cells using viral vectors.

[0169] As used herein, the terms “treatment,” “treating,” or “treat,” with respect to a particular condition, refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse events caused by the disease. When used in this book, "treatment" includes any action of diseases or disability in the subject, especially in humans, and prevents diseases or disorders in the case that is likely to have a disease but still not diagnosed with diseases; Reduces or ease diseases or disorders, that is, to reduce the return of diseases or disability, and to reduce the symptoms of one or more diseases or disability. "Treatment" can also encompass delivery of drugs or administration of therapies to provide a pharmacological effect, even in the absence of a disease, disorder or symptom. The term "treatment" is used in some embodiments to refer to administration of a compound of the disclosure to ameliorate a disease or disorder in a host, preferably a mammalian subject, more preferably a human. Thus, the term "treatment" can include: preventing a disorder from occurring in a host, particularly if the host is susceptible to the disease but has not yet been diagnosed with the disease; inhibiting the disorder; and / or alleviating or reversing the disorder. It is understood that the term "prevent" does not require the disease state to be completely abrogated, so long as the methods of the present disclosure are directed to the prevention of disorders. Rather, as used herein, the term prevent refers to the ability of one skilled in the art to identify populations susceptible to a disorder so that administration of the compounds of the present disclosure can occur prior to the onset of the disease. The term does not imply that the disease state must be completely avoided.

[0170] As used herein, the term "vector" refers to a nucleic acid molecule capable of mediating the entry, eg, translocation, transport, etc. of another nucleic acid molecule into a cell. The nucleic acid to be transferred is typically ligated into, eg, inserted into, the vector nucleic acid molecule. The vector may contain sequences directing autonomous replication or may contain sequences sufficient to allow integration into the host cell DNA. As will be apparent to those skilled in the art, viral vectors can include various viral components in addition to the nucleic acid that mediate entry of the translocating nucleic acid. Many vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphipathic compounds, plasmids and viral vectors. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors (including lentiviral vectors), and the like.

[0171] expression vector The disclosure provides, in some embodiments, expression vectors (eg, lentiviral expression vectors) that include at least one nucleic acid sequence for expression. In some embodiments, the expression vector includes a first nucleic acid sequence that encodes a factor designed to knock down the HPRT1 gene or otherwise result in decreased HPRT1 gene expression. In some embodiments, HPRT1 gene expression is reduced by 80% or more.

[0172] In some embodiments, the disclosure provides an expression vector comprising a first expression control sequence operably linked to a first nucleic acid sequence, wherein the first nucleic acid sequence encodes an shRNA that knocks down hypoxanthine-guanine phosphoribosyltransferase (HPRT), wherein the shRNA has at least 90% identity to the sequence of any one of SEQ ID NOS:2, 5, 6, and 7. In some embodiments, the shRNA has a nucleic acid sequence that is at least 95% identical to the sequence of any one of SEQ ID NOs:2, 5, 6, and 7. In some embodiments, the shRNA has a nucleic acid sequence that is at least 97% identical to the sequence of any one of SEQ ID NOs:2, 5, 6, and 7. In some embodiments, the shRNA comprises the nucleic acid sequence of any one of SEQ ID NOs:2, 5, 6, and 7. In some embodiments, an shRNA that knocks down hypoxanthine-guanine phosphoribosyltransferase (HPRT) is the only transgene for expression in the expression vector.

[0173] In some embodiments, an expression vector is provided that consists essentially of a first expression control sequence operably linked to a first nucleic acid sequence as a transgene for expression, wherein the first nucleic acid sequence encodes an shRNA that knocks down hypoxanthine-guanine phosphoribosyltransferase (HPRT), wherein the shRNA has at least 90% identity to the sequence of any of SEQ ID NOs:2, 5, 6, and 7. In particular, some embodiments provide an expression vector consisting essentially of a first nucleic acid sequence as the only transgene for expression, wherein the first nucleic acid sequence encodes an shRNA that knocks down hypoxanthine-guanine phosphoribosyltransferase (HPRT), wherein the shRNA has at least 90% identity to the sequence of any of SEQ ID NOs:2, 5, 6, and 7.

[0174] In a further aspect, there is provided an expression vector comprising a first expression control sequence operably linked as a transgene to a first nucleic acid sequence, wherein the first nucleic acid sequence encodes an shRNA that knocks down hypoxanthine-guanine phosphoribosyltransferase (HPRT), wherein the shRNA has at least 90% identity to the sequence of any of SEQ ID NOs: 2, 5, 6, and 7, wherein the first nucleic acid sequence is the only element for expression. In particular, some embodiments provide an expression vector comprising a first nucleic acid sequence as the only transgene for expression, wherein the first nucleic acid sequence encodes an shRNA that knocks down hypoxanthine-guanine phosphoribosyltransferase (HPRT), wherein the shRNA has at least 90% identity to the sequence of any of SEQ ID NOs:2, 5, 6, and 7.

[0175] In some embodiments, the expression vector is a self-inactivating lentiviral vector. In other embodiments, the expression vector is a retroviral vector. The lentiviral genome is usually organized into a 5'long terminal repeat (LTR), gag gene, pol gene, env gene, accessory genes (nef, vif, vpr, vpu) and 3'LTR. The viral LTR is divided into three regions called U3, R and U5. The U3 region contains enhancer and promoter elements. The U5 region contains a polyadenylation signal. An R (repeat) region separates the U3 and U5 regions, and transcribed sequences of the R region are found at both the 5' and 3' ends of the viral RNA. For example, "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000)); O Narayan and Clements (1989) J. Gen. Virology, Vol.70: 1617-1639; Fields et al. (1990) Fundamental Virology Raven Press.; See Verma I M. (1998) J Virol., Vol. 72(10):81507, and U.S. Patent No. 6,013,516. Examples of lentiviral vectors that have been used to infect HSCs are described in the following papers, each of which is incorporated herein by reference in its entirety: Evans et al., Hum Gene Ther., Vol. 10:1479-1489, 1999; Case et al., Proc Natl Acad Sci USA, Vol. USA, Vol. 95:11939-11944, 1998; Miyoshi et al., Science Vol. 283:682-686, 1999; and Sutton et al., J. Virol., Vol.

[0176] In some embodiments, the expression vector is a modified lentivirus, thus capable of infecting both dividing and non-dividing cells. In some embodiments, the modified lentiviral genome lacks genes for lentiviral proteins required for viral replication, thus preventing unwanted replication, such as replication in target cells. In some embodiments, proteins required for replication of the modified genome are provided in trans in the packaging cell line during production of the recombinant retrovirus or lentivirus.

[0177] In some embodiments, the expression vector comprises sequences from the lentiviral 5' and 3' long terminal repeats (LTRs). In some embodiments, the vector comprises the R and U5 sequences from the 5'LTR of the lentivirus and the inactivating or self-inactivating 3'LTR from the lentivirus. In some embodiments, the LTR sequence is an HIV LTR sequence.

[0178] Additional components of lentiviral expression vectors (and methods of synthesizing and / or producing such vectors) are disclosed in US Patent Application Publication No. 2018 / 0112220, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the lentiviral expression vector comprises a TL20c backbone that has at least 90% identity to the backbone of SEQ ID NO:16. In some embodiments, the lentiviral expression vector comprises a TL20c backbone that has at least 95% identity to the backbone of SEQ ID NO:16. In some embodiments, the lentiviral expression vector comprises a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO:17. In some embodiments, the lentiviral expression vector comprises a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO:17. In some embodiments, the lentiviral expression vector includes at least one of a WPRE element or a Rev response element (see, eg, SEQ ID NOs: 18 and 19, respectively).

[0179] In some embodiments, the lentiviral vectors discussed herein may be integration-producing or non-integration-producing (also referred to as integration-deficient lentiviruses). As used herein, the term "integration deficient lentivirus" or "IDLV" refers to a lentivirus that has an integrase deficient in the ability to integrate the viral genome into the genome of the host cell. In some applications, the use of lentiviral vectors that cause integration can avoid the possibility of insertional mutagenesis induced by the lentivirus that causes integration. Integration-deficient lentiviral vectors are typically generated by mutating the lentiviral integrase gene or by modifying the junction sequence of the LTR (see, e.g., Sarkis et al., Curr. Gene. Ther., 6:430-437 (2008)). Lentiviral integrase is encoded by the HIV-1 Pol region, which cannot be deleted as it encodes other important activities including reverse transcription, nuclear translocation, and virus particle assembly. Mutations in pol that alter the integrase protein fall into one of two classes: those that selectively affect only integrase activity (class I); or those that have pleiotropic effects (class II). Mutations across the N- and C-termini, as well as the catalytic core region of the integrase protein, generate class II mutations that affect multiple functions, including particle formation and reverse transcription. Class I mutations limit their effects on catalytic activity, DNA binding, linear episomal processing and integrase multimerization. The most common class I mutation sites are a triad of residues in the catalytic core of integrase, including D64, D116, and E152. Each mutation was shown to effectively block integration at up to 4 logs lower frequency of integration than normal, integrating vectors, while maintaining NILV transgene expression. Another alternative method to inhibit integration is to introduce mutations in the integrase DNA binding sites (LTRatt sites) within the 12 base pair region of the U3 region or within the 11 base pair region of the U5 region at the ends of the 5' and 3' LTRs, respectively. These sequences contain a conserved terminal CA dinucleotide that is exposed after integrase-mediated terminal processing. Single or double mutations in the conserved CA / TG dinucleotides result in up to 3-4 log reductions in integration frequency; however, they retain all other necessary functions for effective viral transduction.

[0180] In some embodiments, the vector is an adeno-associated virus (AAV) vector. As used herein, the term "adeno-associated virus (AAV) vector" means an AAV viral particle containing the AAV vector genome, which in turn comprises the first and second expression cassettes referred to herein. It is meant to include AAV vectors of all serotypes, preferably AAV-1 to AAV-9, more preferably AAV-1, AAV-2, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and combinations thereof. AAV vectors resulting from a combination of different serotypes are called hybrid AAV vectors. In one embodiment, the AAV vector is selected from the group consisting of AAV-1, AAV-2, AAV-4, AAV-5, and AAV-6, and combinations thereof. In one embodiment, the AAV vector is an AAV-5 vector. In one embodiment, the AAV vector is an AAV-5 vector containing AAV-2 inverted terminal sequences (ITRs). Also contemplated by this disclosure are AAV vectors comprising variants of naturally occurring viral proteins, such as one or more capsid proteins.

[0181] Ingredients that lead to knockdown of the HPRT1 gene In some embodiments, the nucleic acid sequence encoding a factor designed to knockdown the HPRT1 gene is an RNA interference factor (RNAi). In some embodiments, the RNAi agents are shRNAs, microRNAs, or hybrids thereof.

[0182] RNAi In some embodiments, the expression vector includes a first nucleic acid sequence that encodes RNAi. RNA interference is an approach for post-transcriptional silencing of gene expression by inducing degradation of homologous transcripts by a complex, multistep enzymatic method, such as a method involving sequence-specific double-stranded small interfering RNA (siRNA). A simple model of the RNAi pathway is based on two steps, each involving a ribonuclease enzyme. In the first step, trigger RNAs (either dsRNA or miRNA primary transcripts) are processed into short, interfering RNAs (siRNAs) by the RNaseII enzymes DICER and Drosha. In a second step, siRNAs are loaded into the effector complex RNA-induced silencing complex (RISC). The siRNA is unwound during RISC assembly and single-stranded RNA hybridizes to the mRNA target. Gene silencing is thought to be the result of enzymatic degradation of target mRNAs by the RNaseH enzyme Argonaute (Slicer). If the siRNA / mRNA duplex contains mismatches, the mRNA will not be cleaved. Rather, gene silencing is the result of translational inhibition.

[0183] In some embodiments, the RNAi agent is an inhibitory or silencing nucleic acid. As used herein, "silencing nucleic acid" refers to any polynucleotide capable of interacting with a specific sequence and inhibiting gene expression. Examples of silencing nucleic acids include RNA duplexes (e.g., siRNAs, shRNAs), locked nucleic acids (“LNAs”), antisense RNAs, DNA polynucleotides encoding sense and / or antisense sequences of siRNAs or shRNAs, DNAzymes, or ribozymes. Those skilled in the art will recognize that inhibition of gene expression need not be gene expression from the particular listed sequences, but can be, for example, gene expression from sequences controlled by the particular sequences.

[0184] Methods for constructing interfering RNA are known in the art. For example, the interfering RNA is assembled from two separate oligonucleotides, one strand being the sense strand and the other strand being the antisense strand, the antisense and sense strands being self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to the nucleotide sequence of the other strand; e.g., when the antisense and sense strands form a double or double-stranded structure); Includes nucleotide sequences corresponding to target nucleic acid sequences or portions thereof. Alternatively, the interfering RNA is assembled from a single oligonucleotide and the self-complementary sense and antisense regions are joined by nucleic acid-based or non-nucleic acid-based linkers. An interfering RNA is a polynucleotide with a double-stranded, asymmetric duplex, hairpin, or asymmetric hairpin secondary structure having self-complementary sense and antisense regions, the antisense region comprising a nucleotide sequence complementary to the nucleotide sequence of a separate target nucleic acid molecule or portion thereof, and the sense region having a nucleotide sequence corresponding to the target nucleic acid sequence or portion thereof. An interfering RNA is a circular single-stranded polynucleotide having a stem comprising two or more loop structures and self-complementary sense and antisense regions, the antisense region comprising a nucleotide sequence complementary to a nucleotide sequence of a target nucleic acid molecule or portion thereof, the sense region comprising a nucleotide sequence corresponding to the target nucleic acid sequence or portion thereof, and the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.

[0185] In some embodiments, an interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region. When expressed, such RNA molecules form the desired "hairpin" structure and are referred to herein as "shRNAs." In some embodiments, the loop region is typically between about 2 and about 10 nucleotides in length (see SEQ ID NO:20, by way of example only). In other embodiments, the loop region is about 6 to about 9 nucleotides long. In some embodiments, the sense and antisense regions are between about 15 and about 30 nucleotides in length. After post-transcriptional processing, small hairpin RNAs are converted into siRNAs by cleavage events mediated by the enzyme DICER, a member of the RNaseIII family. The siRNA can then inhibit the expression of genes with which it shares homology. For further details, see Brummelkamp et al., Science 296:550-553, (2002); Lee et al., Nature Biotechnol., 20, 500-505, (2002); Miyagishi and Taira, Nature Biotechnol 20:497-500, (2002); ison et al., Genes & Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002); Sui, Proc. Natl. Acad. 6052, (2002).

[0186] shRNA In some embodiments, the first nucleic acid sequence encodes an shRNA that targets the HPRT1 gene. In some embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 90% identity to the nucleic acid sequence of SEQ ID NO: 1 (referred to herein as "sh734"). In still other embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 95% identity to the nucleic acid sequence of SEQ ID NO:1. In a further embodiment, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 96% identity to the nucleic acid sequence of SEQ ID NO:1. In a further embodiment, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 97% identity to the nucleic acid sequence of SEQ ID NO:1. In an even further embodiment, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 98% identity to the nucleic acid sequence of SEQ ID NO:1. In an even further embodiment, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 99% identity to the nucleic acid sequence of SEQ ID NO:1. In other embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has the nucleic acid sequence of SEQ ID NO:1.

[0187] In some embodiments, the nucleic acid sequence of SEQ ID NO: 1 is modified. In some embodiments, the modifications are: (i) incorporation of a hsa-miR-22 loop sequence (e.g., CCUGACCCA) (SEQ ID NO:21); (ii) addition of a 5' to 3' nucleotide spacer, such as a spacer with 2 or 3 nucleotides (e.g., TA); (iii) modification of the 5' start, such as addition of one or more nucleotides (e.g., G); and / or (iv) addition of two nucleotides 5' and 3' to the stem and loop (e.g., 5'A and 3' T). In general, first-generation shRNAs are processed into a heterogeneous mixture of small RNAs, and accumulation of precursor transcripts has been shown to induce both sequence-dependent and independent non-specific off-target effects in vivo. Therefore, based on the current understanding of DICER processing and specificity, design rules were applied to a design that would optimize sh734 structure and DICER processing capacity and efficacy (Gu, S., Y. Zhang, L. Jin, Y. Huang, F. Zhang, M. C. Bassik, M. Kampmann, and M. A. Kay. 2014. Weak base pairing in both seed and 3'regions reduce RNAi off-targets and See also enhances si / shRNA designs. Nucleic Acids Research 42:12169-12176).

[0188] In some embodiments, the nucleic acid sequence of SEQ ID NO:1 is modified by adding two nucleotides 5' and 3' (e.g., G and C, respectively) to the hairpin loop (SEQ ID NO:20), thereby extending the guide strand from about 19 nucleotides in length to about 21 nucleotides in length, and replacing the loop with the hsa-miR-22 loop CCUGACCCA (SEQ ID NO:21) to provide the nucleotide sequence of SEQ ID NO:2. In some embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 90% identity to the nucleic acid sequence of SEQ ID NO:2. In other embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 95% identity to the nucleic acid sequence of SEQ ID NO:2. In other embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 96% identity to the nucleic acid sequence of SEQ ID NO:2. In other embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 97% identity to the nucleic acid sequence of SEQ ID NO:2. In other embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 98% identity to the nucleic acid sequence of SEQ ID NO:2. In other embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 99% identity to the nucleic acid sequence of SEQ ID NO:2. In still other embodiments, the nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has the sequence of SEQ ID NO:2. The shRNA encoded by SEQ ID NO:2 appears to achieve similar knockdown of HPRT compared to SEQ ID NO:1. Similarly, cells substantially deficient in HPRT through knockdown of HPRT by expression of shRNA encoded by SEQ ID NO:2 would allow selection using thioguanine analogues (eg, 6-TG or 6-MP).

[0189] In some embodiments, the RNAi present in the vector encodes a nucleic acid molecule, such as a nucleic acid molecule having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the RNAi present in the vector encodes a nucleic acid molecule, such as a nucleic acid molecule having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, nucleic acid molecules having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4 are found in the cytoplasm of the host cell.

[0190] In some embodiments, the present disclosure provides host cells comprising at least one nucleic acid molecule having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the present disclosure provides host cells comprising at least one nucleic acid molecule having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the present disclosure provides host cells comprising at least one nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0191] In some embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 80% identity to the nucleic acid sequence of SEQ ID NO:5 (referred to herein as "shHPRT616"). In other embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 90% identity to the nucleic acid sequence of SEQ ID NO:5. In still other embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 95% identity to the nucleic acid sequence of SEQ ID NO:5. In a further embodiment, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 96% identity to the nucleic acid sequence of SEQ ID NO:5. In a further embodiment, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 97% identity to the nucleic acid sequence of SEQ ID NO:5. In an even further embodiment, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 98% identity to the nucleic acid sequence of SEQ ID NO:5. In still further embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 99% identity to the nucleic acid sequence of SEQ ID NO:5. In another embodiment, the nucleic acid sequence encoding the shRNA targeting the HPRT1 gene has the sequence of SEQ ID NO:5 (see also Figure 3).

[0192] In some embodiments, the first nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 80% identity to the nucleic acid sequence of SEQ ID NO: 6 (referred to herein as "shHPRT211"). In other embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 90% identity to the nucleic acid sequence of SEQ ID NO:6. In still other embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 95% identity to the nucleic acid sequence of SEQ ID NO:6. In a further embodiment, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 96% identity to the nucleic acid sequence of SEQ ID NO:6. In a further embodiment, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 97% identity to the nucleic acid sequence of SEQ ID NO:6. In still further embodiments, the nucleic acid sequence encoding the shRNA that targets the HPRT1 gene has a sequence that has at least 98% identity to the nucleic acid sequence of SEQ ID NO:6. In still further embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 99% identity to the nucleic acid sequence of SEQ ID NO:6. In another embodiment, the nucleic acid sequence encoding the shRNA targeting the HPRT1 gene has the sequence of SEQ ID NO: 6 (see also Figure 4).

[0193] In some embodiments, the nucleic acid sequence encoding the shRNA that targets the HPRT1 gene has a sequence that has at least 80% identity to the nucleic acid sequence of SEQ ID NO: 7 (referred to herein as "shHPRT734.1") (see also Figure 5). In other embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 90% identity to the nucleic acid sequence of SEQ ID NO:7. In still other embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 95% identity to the nucleic acid sequence of SEQ ID NO:7. In a further embodiment, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 96% identity to the nucleic acid sequence of SEQ ID NO:7. In a further embodiment, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 97% identity to the nucleic acid sequence of SEQ ID NO:7. In an even further embodiment, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 98% identity to the nucleic acid sequence of SEQ ID NO:7. In still further embodiments, a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene has a sequence that has at least 99% identity to the nucleic acid sequence of SEQ ID NO:7. In another embodiment, the nucleic acid sequence encoding the shRNA targeting the HPRT1 gene has the sequence of SEQ ID NO:7 (see also Figure 5).

[0194] micro RNA MicroRNAs (miRs) are a group of non-coding RNAs that post-transcriptionally regulate the expression of their target genes. These single-stranded molecules form miRNA-mediated silencing complexes (miRISC) complexes with other proteins that bind to the 3' untranslated regions (UTRs) of their target mRNAs to prevent their translation in the cytoplasm.

[0195] In some embodiments, shRNA sequences are embedded in microRNA secondary structures (“microRNA-based shRNAs”). In some embodiments, the HPRT-targeting shRNA nucleic acid sequences are embedded in microRNA secondary structures. In some embodiments, microRNA-based shRNAs target coding sequences within HPRT to achieve knockdown of HPRT expression, which would be equivalent to saturation of the concomitant pathway and utilization of shRNAs that target HPRT without cytotoxicity or off-target effects. In some embodiments, the microRNA-based shRNA is a novel artificial microRNA shRNA. The production of such novel microRNA-based shRNAs is described in Fang, W. & Bartel, David P. The Menu of Features that Define Primary MicroRNAs and Enable De Novo Design of MicroRNA Genes. Molecular Cell 60, 131-145, the disclosure of which is incorporated herein by reference in its entirety.

[0196] In some embodiments, the microRNA-based shRNA has a nucleic acid sequence that has at least 80% identity with the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the microRNA-based shRNA has a nucleic acid sequence that has at least 90% identity with the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the microRNA-based shRNA has a nucleic acid sequence with at least 95% identity to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the microRNA-based shRNA has a nucleic acid sequence that has at least 96% identity to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the microRNA-based shRNA has a nucleic acid sequence that has at least 97% identity to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the microRNA-based shRNA has a nucleic acid sequence that has at least 98% identity to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the microRNA-based shRNA has a nucleic acid sequence that has at least 99% identity to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the microRNA-based shRNA has the sequence of SEQ ID NO:8 (“miRNA734-Denovo”) (see also Figure 6). The RNA form of SEQ ID NO:8 has SEQ ID NO:22.

[0197] In some embodiments, the microRNA-based shRNA has a sequence that has at least 80% identity with the sequence of SEQ ID NO:9. In some embodiments, the microRNA-based shRNA has a nucleic acid sequence that has at least 90% identity with the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the microRNA-based shRNA has a sequence that has at least 95% identity to the sequence of SEQ ID NO:9. In some embodiments, the microRNA-based shRNA has a sequence that has at least 96% identity to the sequence of SEQ ID NO:9. In some embodiments, the microRNA-based shRNA has a sequence that has at least 97% identity to the sequence of SEQ ID NO:9. In some embodiments, the microRNA-based shRNA has a sequence that has at least 98% identity to the sequence of SEQ ID NO:9. In some embodiments, the microRNA-based shRNA has a sequence that is at least 99% identical to the sequence of SEQ ID NO:9. In some embodiments, the microRNA-based shRNA has the nucleic acid sequence of SEQ ID NO: 9 (“miRNA211-Denovo”) (see also Figure 7). The RNA form of SEQ ID NO:9 has SEQ ID NO:23.

[0198] In other embodiments, the microRNA-based shRNA is the third generation miRNA scaffold-modified miRNA16-2 (hereinafter "miRNA-3G") (see, eg, Figures 8 and 9). The synthesis of such miRNA-3G molecules is described in Watanabe, C., Cuellar, T.L. & Haley, B. “Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi,” RNA Biology 13, 25-33 (2016), the disclosure of which is incorporated herein by reference in its entirety.

[0199] In some embodiments, miRNA-3G has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO:10. In some embodiments, miRNA-3G has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:10. In some embodiments, miRNA-3G has a sequence that has at least 95% identity to the sequence of SEQ ID NO:10. In some embodiments, miRNA-3G has a sequence that has at least 96% identity to the sequence of SEQ ID NO:10. In some embodiments, the miRNA-3G has a sequence with at least 97% identity to the sequence of SEQ ID NO:10. In some embodiments, miRNA-3G has a sequence that has at least 98% identity to the sequence of SEQ ID NO:10. In some embodiments, the miRNA-3G has a sequence that has at least 99% identity to the sequence of SEQ ID NO:10. In some embodiments, miRNA-3G has the nucleic acid sequence of SEQ ID NO: 10 (“miRNA211-3G”) (see also Figure 9).

[0200] In some embodiments, miRNA-3G has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO:11. In some embodiments, miRNA-3G has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:11. In some embodiments, miRNA-3G has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the miRNA-3G has a nucleic acid sequence with at least 96% identity to the nucleic acid sequence of SEQ ID NO:11. In some embodiments, miRNA-3G has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:11. In some embodiments, miRNA-3G has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:11. In some embodiments, miRNA-3G has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:11. In other embodiments, miRNA-3G has the nucleic acid sequence of SEQ ID NO: 11 (“miRNA734-3G”) (see also Figure 8).

[0201] In some embodiments, sh734shRNA is adapted to mimic miRNA-451 (see SEQ ID NO:24) structure with a 17-nucleotide base pair stem and a 4-nucleotide loop (miR-451 regulates the drug transport protein P-glycoprotein). Notably, this structure does not require processing by DICER. The pre-451 mRNA structure is thought to be cleaved by Ago2 followed by poly(A)-specific ribonuclease (PARN) to generate the mature miRNA-451 structural mimic. Ago-shRNA mimics the structure of endogenous miR-451 and may have the advantage of being DICER-independent. This is thought to limit off-target effects of passenger loading due to variable 3'-5' exonuclease activity (mature 23-26 nt) (See Herrera-Carrillo, E., and B. Berkhout. 2017. DICER-independent processing of small RNA duplexes: mechanistic insights and applications. Nucleic Acids Res. 45:10369-10379). There may also be advantages to exploiting alternative DICER-independent processing of shRNAs, including effective reduction of off-target effects of single RNAi activity guides, lack of saturation of the cellular RNAi DICER machinery, and shorter RNA duplexes are less likely to elicit innate RIG-I responses.

[0202] Alternative to RNAi As an alternative to incorporating RNAi, in some embodiments, an expression vector may contain a nucleic acid sequence encoding an antisense oligonucleotide that binds to a site of messenger RNA (mRNA). Antisense oligonucleotides of the disclosure specifically hybridize to nucleic acids encoding proteins and interfere with protein transcription or translation. In some embodiments, antisense oligonucleotides target DNA and interfere with its replication and / or transcription. In other embodiments, antisense oligonucleotides specifically hybridize to pre-mRNA (ie, precursor mRNA, which is an immature single strand of mRNA), and RNA, including mRNA. Such antisense oligonucleotides can affect, for example, translocation of RNA to the site of protein translation, translation of protein from RNA, splicing of RNA to produce one or more mRNA species, and catalytic activity involving or facilitated by RNA. The overall effect of such interference is to modulate, reduce or inhibit target protein expression.

[0203] In some embodiments, the expression vector incorporates a nucleic acid sequence encoding an exon-skipping factor or exon-skipping transgene. As used herein, the phrase "exon-skipping transgene" or "exon-skipping factor" refers to any nucleic acid encoding an antisense oligonucleotide capable of producing exon-skipping. "Exon skipping" refers to exons that are skipped and removed at the pre-mRNA level during protein production. Antisense oligonucleotides could interfere with splice sites or regulatory elements within exons. This can result in a truncated, partially functional protein despite the presence of genetic mutations. In general, antisense oligonucleotides may be mutation-specific and can bind to mutation sites in pre-messenger RNA and induce exon skipping.

[0204] Exon-skipping transgenes encode factors that can cause exon skipping, such factors being antisense oligonucleotides. Antisense oligonucleotides can interfere with splice sites or regulatory elements within exons, resulting in truncated, partially functional proteins despite the presence of genetic mutations. In addition, antisense oligonucleotides can be mutation-specific, binding to mutation sites in pre-messenger RNA and inducing exon skipping. Antisense oligonucleotides for exon skipping are known in the art and commonly referred to as AONs. Such AONs include small nuclear RNAs (“snRNAs”), which is a class of small RNA molecules that are confined to the nucleus and participate in splicing or other RNA processing reactions. Examples of antisense oligonucleotides, methods of designing them, and related methods of production, e.g. 76, 20140350067 and 20140329762.

[0205] In some embodiments, an expression vector of the disclosure comprises a nucleic acid encoding an exon-skipping factor that results in exon skipping during expression of HPRT or results in HPRT duplication mutations (e.g., duplication mutations in exon 4) (Baba S et al. Novel mutation in HPRT1 causing a splicing error with multiple variations. Nucleosides Nucleotides Nucleic Acids. Jan. 2017 2;36(1), the disclosure of which is incorporated herein by reference in its entirety). : see pages 1-6).

[0206] In some embodiments, HPRT can be replaced by spliceosomal trans-splicing with an altered mutated sequence, thus promoting HPRT. In some embodiments, this requires (1) a mutated coding region that replaces the coding sequence in the target RNA, (2) a 5' or 3' splice site, and / or (3) a binding domain, i.e., an antisense oligonucleotide sequence that is complementary to the target HPRT RNA. In some embodiments all three elements are required.

[0207] promoter A variety of promoters are used to drive expression of each of the nucleic acid sequences incorporated into the disclosed expression vectors. For example, a first nucleic acid sequence encoding RNAi (eg, an anti-HPRT shRNA) is expressed from a first promoter selected from one of a Pol III promoter or a Pol II promoter. Similarly, by another example, a first nucleic acid sequence encoding a microRNA-based shRNA that downregulates HPRT is expressed from a first promoter selected from one of a Pol III promoter or a Pol II promoter. In some embodiments, the promoter may be a constitutive promoter or an inducible promoter known to those skilled in the art. In some embodiments, the promoter comprises at least a portion of an HIV LTR (eg TAR).

[0208] Non-limiting examples of suitable promoters include, but are not limited to, RNA polymerase I (pol I), polymerase II (pol II), or polymerase III (pol III) promoters. An "RNA polymerase III promoter" or "RNA pol III promoter" or "polymerase III promoter" or "pol III promoter" is any invertebrate, vertebrate, or mammalian, e.g., human, mouse, porcine, bovine, primate, monkey, or the like promoter that associates or interacts with RNA polymerase III in its natural context in a cell to transcribe its operably linked gene, or a promoter that interacts with RNA polymerase III to transcribe its operably linked nucleic acid sequence in a host cell of choice, or one that has been engineered, whether in nature. and any variant thereof. RNA pol III promoters suitable for use in the expression vectors of the disclosure include, but are not limited to, human U6, mouse U6, human H1, and others.

[0209] Examples of pol II promoters include, but are not limited to Ef1alpha, CMV, and ubiquitin. Other specific pol II promoters include the ankyrin promoter (Sabatino DE, et al., Proc Natl Acad Sd USA. (24):13294-9 (2000)), the spectrin promoter (Gallagher PG, et al., J Biol Chem. 274(10):6062-73, (2000)), the transferrin receptor promoter (Marziali G, et al., Oncogene.21(52):7 933-44, (2002)), band 3 / anion transporter promoter (Frazar TF, et al., MoI Cell Biol (14):4753-63, (2003)), band 4.1 promoter (Harrison PR, et al., Exp Cell Res. 155(2):321-44, (1984)), BcI-Xl promoter (Tian C, et al., Blood 15;101). (6): 2235-42 (2003)), EKLF promoter (Xue L, et al., Blood. 103(11): 4078-83 (2004)).Epub Feb. 5, 2004), ADD2 promoter (Yenerel MN, et al., Exp Hematol. 33(7): 758-66 (2005)), DYRK3 promoter (Zhang D, et al., Genomics 85(1):117-30 (2005)), SOCS promoter (Sarna MK, et al., Oncogene 22(21):3221-30 (2003)), LAF promoter (To MD, et al., bit J Cancer l;115(4):568-74, (2005)), PSMA promoter (Zeng H, et al., JAndrol(2):215-21). , (2005)), PSA promoter (Li HW, et al., Biochem Biophys Res Commun 334(4):1287-91, (2005)), probasin promoter (Zhang J, et al., 145(l):134-48, (2004)). ancer Res. 18(3A): 1357-60, (1998)), synapsin promoter (Thiel G, et al., ProcNatl Acad Sd USA., 88(8): 3431-5 (1988)), Willebrand factor promoter (Jahroudi N, Lynch DC. MoI Cell-5zo / .14(2): 999-1008, (1994)), FLTl ( Nicklin SA, et al., Hypertension 38(l):65-70, (2001)), Tau promoter (Sadot E, et al., JMoI Biol. 256(5):805-12, (1996)), Tyrosinase promoter (Lillehammer T, et al., Cancer Gene Ther. (2005)), Pander promoter (Burkhardt BR, et al., Biochim Biophys Acta). (2005)), neuro-specific enolase promoter (Levy YS, et al., JMolNeurosci. 21(2):121-32, (2003)), hTERT promoter (Ito H, et al., Hum Gene Ther 16(6):685-98, (2005)), HRE response element (Chadderton N, et al., IntJRadiat Oncol Biol Phys. 62( l): 2U-22, (2005)), the lck promoter (Zhang DJ, et al., J Immunol. 174(11):6725-31, (2005)), the MHCII promoter (De Geest BR, et al., Blood. 101(7): 2551-6, (2003), Epub November 21, 2002), and the CDl Ic promoter (Lopez -Rodriguez C, et al., J Biol Chem. 272(46):29120-6 (1997)).

[0210] In some embodiments, the promoter driving expression of the agent designed to knockdown HPRT is the RNA pol III promoter. In some embodiments, the promoter driving expression of an agent designed to knockdown HPRT is the 7sk promoter (eg, 7SK human 7SK RNA promoter). In some embodiments, the 7sk promoter has the nucleic acid sequence provided by accession AY578685 (Homo sapiens cell line HEK-293 7SK RNA promoter region, complete sequence, accession AY578685).

[0211] In some embodiments, the 7sk promoter has a sequence that has at least 90% identity to the sequence of SEQ ID NO:14. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 95% identity to the sequence of SEQ ID NO:14. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 96% identity to the sequence of SEQ ID NO:14. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 97% identity to the sequence of SEQ ID NO:14. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 98% identity to the sequence of SEQ ID NO:14. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 99% identity to the sequence of SEQ ID NO:14. In some embodiments, the 7sk promoter has the nucleic acid sequence shown in SEQ ID NO:14.

[0212] In some embodiments, the 7sk promoter utilized contains at least one mutation and / or deletion in its nucleic acid sequence relative to the 7sk promoter. Suitable 7sk promoter mutations are described in Boyd, D.C., Turner, P.C., Watkins, N.J., Gerster, T. & Murphy, S. Functional Redundancy of Promoter Elements Ensures Efficient Transcription of the Human 7SK Gene in vivo. Journal of Molecular Biology 253, 677-690 (1995), the disclosure of which is hereby incorporated by reference. incorporated herein in its entirety. In some embodiments, functional mutations or deletions are made in cis-regulatory elements in the 7sk promoter to regulate the expression level of promoter-driven transgenes, including sh734. The mutations described are used to establish a correlation between sh734 expression levels driven by the Pol III promoter, introducing functionality that undergoes stable selection and long-term stability and safety in the presence of 6-TG and / or 6-MP therapy. The location of the 7sk promoter mutation is depicted in FIG.

[0213] In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 95% identity to the sequence of SEQ ID NO:15. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 96% identity to the sequence of SEQ ID NO:15. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 97% identity to the sequence of SEQ ID NO:15. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 98% identity to the sequence of SEQ ID NO:15. In some embodiments, the 7sk promoter has a nucleic acid sequence with at least 99% identity to the sequence of SEQ ID NO:15. In some embodiments, the 7sk promoter has the nucleic acid sequence shown in SEQ ID NO:15.

[0214] In other embodiments, the promoter is a tissue-specific promoter. Some non-limiting examples of tissue-specific promoters that may be used include lck (see, e.g., Garvin et al., MoI. Cell Biol. 8:3058-3064, (1988) and Takadera et al., MoI. Cell Biol. 9:2173-2180, (1989)), myogenin (Yee et al., Genes and Development 7:1277-1289 (1993)). , and thyl (Gundersen et al., Gene 113:207-214, (1992)).

[0215] Non-limiting examples of combinations of nucleic acid sequences operably linked to promoters are shown in the table below.

[0216]

table 1

[0217] Vector production In some embodiments, an expression cassette, such as a cassette comprising a nucleic acid sequence adapted to knockdown HPRT, is inserted into an expression vector, such as a lentiviral expression vector, to provide the vector with at least one transgene for expression. In some embodiments, the lentiviral expression vector is pTL20c, pTL20d, FG, pRRL, pCL20, pLKO.1 puro, pLKO.1, pLKO.3G, Tet-pLKO-puro, pSico, pLJM1-EGFP, FUGW, pLVTHM, pLVUT-tTR-KRAB, pLL3.7, pLB, pWPXL, pWPI, EF.CMV.R FP, pLenti CMV Puro DEST, pLenti-puro, pLOVE, pULTRA, pLJM1-EGFP, pLX301, pInducer20, pHIV-EGFP, Tet-pLKO-neo, pLV-mCherry, pCW57.1, pLionII, pSLIK-Hygro, and pInducer10-mir-RUP-PheS. In other embodiments, the lentiviral expression vector is AnkT9W vector, T9Ank2W vector, TNS9 vector, lentiglobin HPV569 vector, lentiglobin BB305 vector, BG-1 vector, BGM-1 vector, d432βAγ vector, mLAβΔγV5 vector, GLOBE vector, G-GLOBE vector, βAS3-FB vector, V5 vector, V5m3 vector, V5m3-400 vector, G9 vectors, and BCL11A shmir vectors can be selected. In some embodiments, the lentiviral expression vector may be selected from the group consisting of pTL20c, pTL20d, FG, pRRL and pCL20. In still other embodiments, the lentiviral expression vector is pTL20c.

[0218] In some embodiments, the expression cassette comprises a nucleic acid sequence having at least 95% identity to the sequence of SEQ ID NO:13. In other embodiments, the expression cassette comprises a nucleic acid sequence having at least 96% identity to the sequence of SEQ ID NO:13. In other embodiments, the expression cassette comprises a nucleic acid sequence having at least 97% identity to the sequence of SEQ ID NO:13. In other embodiments, the expression cassette comprises a nucleic acid sequence having at least 98% identity to the sequence of SEQ ID NO:13. In still other embodiments, the expression cassette comprises a nucleic acid sequence having at least 99% identity to the sequence of SEQ ID NO:13. In a further embodiment, the expression cassette has the nucleic acid sequence of SEQ ID NO:13.

[0219] In some embodiments, the plasmid has a nucleic acid sequence with at least 90% identity to SEQ ID NO:17. In some embodiments, the plasmid has a nucleic acid sequence with at least 95% identity to SEQ ID NO:17. In some embodiments, the plasmid has a nucleic acid sequence with at least 96% identity to SEQ ID NO:17. In some embodiments, the plasmid has a nucleic acid sequence with at least 97% identity to SEQ ID NO:17. In some embodiments, the plasmid has a nucleic acid sequence with at least 98% identity to SEQ ID NO:17. In some embodiments, the plasmid has a nucleic acid sequence with at least 99% identity to SEQ ID NO:17. In some embodiments, the plasmid has the nucleic acid sequence of SEQ ID NO:17.

[0220] In some embodiments, the plasmid comprises a TL20 viral backbone having a nucleic acid sequence with at least 90% identity to the sequence of SEQ ID NO:16. In some embodiments, the plasmid comprises a TL20 viral backbone having a nucleic acid sequence with at least 95% identity to the sequence of SEQ ID NO:16. In some embodiments, the plasmid comprises a TL20 viral backbone having a nucleic acid sequence with at least 96% identity to the sequence of SEQ ID NO:16. In some embodiments, the plasmid comprises a TL20 viral backbone having a nucleic acid sequence with at least 97% identity to the sequence of SEQ ID NO:16. In some embodiments, the plasmid comprises a TL20 viral backbone having a nucleic acid sequence with at least 98% identity to the sequence of SEQ ID NO:16. In some embodiments, the plasmid comprises a TL20 viral backbone having a nucleic acid sequence with at least 99% identity to the sequence of SEQ ID NO:16. In some embodiments, the plasmid comprises a TL20 viral backbone having the nucleic acid sequence of SEQ ID NO:16.

[0221] In one or more embodiments, the first nucleic acid sequence encoding the shRNA that targets the HPRT1 gene is inserted in the expression vector in a different orientation relative to the other vector elements (e.g., compare the orientation of the 7sk promoter between Figures 32). For example, the 7sk-driven sh734 element may be oriented in the same or opposite direction when compared to a transgene such as the UbC-driven GFP described in the Examples. In still other embodiments, the first nucleic acid sequence encoding the shRNA targeting the HPRT1 gene may be inserted at a different location in the expression vector, i.e., upstream or downstream of other vector elements, e.g., upstream or downstream of UbC-driven GFP. It is believed that different positions and / or orientations of the 7sk expression cassette relative to other vector elements may enhance expression of sh734.

[0222] In some embodiments, the 7sk / sh734 expression cassette is located upstream to other vector elements, such as UbC-driven GFP.

[0223] In some embodiments, the 7sk / sh734 expression cassette is located downstream to other vector elements, such as UbC-driven GFP.

[0224] In some embodiments, the 7sk / sh734 expression cassette and other vector elements such as UbC-driven GFP are oriented in the same direction.

[0225] In some embodiments, the 7sk / sh734 expression cassette and other vector elements, such as UbC-driven GFP, are oriented in opposite directions.

[0226] In some embodiments, the 7sk / sh734 expression cassette is oriented in the forward orientation relative to other vector elements such as UbC-driven GFP.

[0227] In some embodiments, the 7sk / sh734 expression cassette is oriented in reverse orientation with respect to other vector elements, such as UbC-driven GFP.

[0228] In some embodiments, the 7sk / sh734 expression cassette is located upstream and oriented in the forward direction relative to other vector elements, such as UbC-driven GFP.

[0229] In some embodiments, the 7sk / sh734 expression cassette is located upstream and oriented in reverse orientation relative to other vector elements, such as UbC-driven GFP.

[0230] In some embodiments, the 7sk / sh734 expression cassette is positioned downstream and oriented in the forward direction relative to other vector elements, such as UbC-driven GFP.

[0231] In some embodiments, the 7sk / sh734 expression cassette is located downstream and oriented in reverse orientation relative to other vector elements, such as UbC-driven GFP.

[0232] Physical and non-viral delivery of agents that downregulate HPRT or knock out HPRT

[0233] In some embodiments, agents designed to knockdown or knockout the HPRT1 gene (including expression constructs containing RNAi) are delivered through physical methods. In some embodiments the physical method is selected from microinjection and electroporation. Electroporation is a technique for introducing chemicals, small molecules, proteins, nucleic acids, etc. into cells by applying an electric field to cells to increase the permeability of cell membranes. Microinjection is a technique for introducing chemicals, small molecules, proteins, nucleic acids, etc. into a single cell by inserting a micropipette into the target cell. Microinjection provides controlled delivery volume and targeted delivery to intracellular locations.

[0234] In some embodiments, (i) an endonuclease and (ii) a guide RNA molecule targeting one sequence in exon 3 or exon 8 of the HPRT1 gene is introduced into lymphocytes by electroporation or microinjection. In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 91% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 92% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 93% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 94% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 95% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 96% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA comprises the sequence of any one of SEQ ID NOs:40-44 and 46-56.

[0235] In some embodiments, (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence in exon 2 of the HPRT1 gene is introduced into lymphocytes by electroporation or microinjection. In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 91% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 92% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 93% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 94% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 96% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA comprises the sequence of any one of SEQ ID NOs:45 and 57-61.

[0236] In some embodiments, the endonuclease delivered by one or more physical methods comprises a Cas protein. In some embodiments the Cas protein comprises a Cas9 protein. In some embodiments, the Cas protein comprises Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0237] In some embodiments, agents designed to knockdown or knockout the HPRT1 gene (including expression constructs containing RNAi) are delivered through non-viral delivery vehicles. In some embodiments, the non-viral delivery vehicle is a nanocapsule or other non-viral delivery vehicle. In some embodiments, (i) an endonuclease and (ii) a guide RNA molecule targeting one sequence in exon 3 or exon 8 of the HPRT1 gene are introduced into lymphocytes by non-viral delivery vehicles such as nanocapsules. In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 95% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:40-44 and 46-56. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:40-44 and 46-56.

[0238] In some embodiments, (i) an endonuclease and (ii) a guide RNA molecule targeting a sequence in exon 2 of the HPRT1 gene are introduced into lymphocytes by non-viral delivery vehicles such as nanocapsules. In some embodiments, the guide RNA molecule has at least 90% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 95% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 97% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 98% sequence identity with any one of SEQ ID NOs:45 and 57-61. In some embodiments, the guide RNA molecule has at least 99% sequence identity with any one of SEQ ID NOs:45 and 57-61.

[0239] In some embodiments, the endonuclease delivered by the non-viral delivery vehicle comprises a Cas protein. In some embodiments the Cas protein comprises a Cas9 protein. In some embodiments, the Cas protein comprises Cas12 protein. In some embodiments, the Cas12 protein is the Cas12a protein. In some embodiments, the Cas12 protein is the Cas12b protein.

[0240] Physical delivery or delivery of agents through non-viral delivery vehicles represents an alternative to achieve down-regulation of HPRT (eg, HPRT1) by expressed RNAi or other agents from expression vectors. As further described herein, it is possible to deliver the gene editing machinery using one or more physical methods such as antisense RNA, oligonucleotides designed for exon skipping, or nanocapsules or electroporation.

[0241] In general, nanocapsules are vesicle systems exhibiting a typical core-shell structure in which the active molecules are confined in reservoirs or cavities surrounded by a polymeric membrane or coating. In some embodiments, a typical nanocapsule shell is made of a polymer film or coating. In some embodiments, the nanocapsules are derived from biodegradable or bioerodible polymeric materials, ie, the nanocapsules are biodegradable and / or erodible polymeric nanocapsules. For example, components for knockdown and / or knockout are encapsulated within nanocapsules comprising one or more biodegradable polymers such as polylactic-polyglycolide, poly(orthoesters) and poly(anhydrides). In some embodiments, polymeric nanocapsules comprise two different positively charged monomers, at least one neutral monomer, and a crosslinker. In some embodiments, the nanocapsules are enzymatically degradable nanocapsules. In some embodiments, nanocapsules consist of a single protein core and a thin polymer shell crosslinked by peptides. In some embodiments, the nanocapsule may be selected such that it can be specifically recognized and cleaved by proteases. In some embodiments, a cleavable cross-linker comprises a peptide sequence or structure that is a substrate for a protease or another enzyme.

[0242] Examples of nanocapsules include, but are not limited to, those described in U.S. Patent Application No. 9,782,357; those described in U.S. Patent Application Publication Nos. 2017 / 0354613 and U.S. Patent Application Publication Nos. 2015 / 0071999; and those described in WO 2016 / 085808 and WO 2017 / 205541. The disclosure of said patent document is hereby incorporated herein by reference in its entirety. In some embodiments, the nanocapsules described in the aforementioned publications may be modified to transport and / or encapsulate components for knockdown and / or knockout, such as Cas proteins and / or gRNAs. Other suitable nanocapsules, methods of their synthesis, and / or encapsulation methods are further disclosed in US Patent Application Publication No. 2011 / 0274682, the disclosure of which is hereby incorporated by reference in its entirety. Still other suitable nanocapsules that may be modified to transport and / or encapsulate components to achieve knockdown or knockout of HPRT are described in WO2013 / 138783, WO2013 / 033717, and WO2014 / 093966, the disclosures of which are hereby incorporated by reference in their entireties.

[0243] In some embodiments, nanocapsules are adapted to target specific cell types (eg, T cells, CD34 hematopoietic stem and progenitor cells) in vivo. For example, a nanocapsule may contain one or more targeting moieties attached to a polymeric nanocapsule. In some embodiments, the targeting moiety delivers the polymeric nanocapsule to a specific cell type, wherein the cell type is selected from the group comprising immune cells, blood cells, heart cells, lung cells, visual cells, liver cells, kidney cells, brain cells, cells of the central nervous system, cells of the peripheral nervous system, cancer cells, virally infected cells, stem cells, skin cells, intestinal cells, and / or auditory cells. In some embodiments, targeting moieties are antibodies.

[0244] In some embodiments, the nanocapsules further comprise at least one targeting moiety. In some embodiments, the nanocapsules comprise between 2-6 targeting moieties. In some embodiments, targeting moieties are antibodies. In some embodiments, the targeting moiety targets any one of the CD117, CD10, CD34, CD38, CD45, CD123, CD127, CD135, CD44, CD47, CD96, CD2, CD4, CD3, and CD9 markers. In some embodiments, the targeting moiety targets any one of the human mesenchymal stem cell CD markers, including the CD29, CD44, CD90, CD49a-f, CD51, CD73(SH3), CD105(SH2), CD106, CD166, and Stro-1 markers. In some embodiments, the targeting moiety targets any one of human hematopoietic stem cells, including CD34, CD38, CD45RA, CD90, and CD49.

[0245] In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127 or FoxP3 and CD44. In some embodiments, the T cell marker is CD3.In some embodiments, the T cell marker is CD28.

[0246] In some embodiments, costimulation by one or more costimulatory moieties can be used to activate target cells, including T cells. In some embodiments, co-stimulation can be achieved by activating one or more cell surface markers, including but not limited to CD28, ICOS, CTLA4, PD1, PD1H, and BTLA. In some embodiments, the co-stimulatory moiety is an antibody.

[0247] Those skilled in the art understand that immune cells can rely on secondary signals to activate an immune response (ie costimulation). For example, T cells require two stimuli to fully activate an immune response. In some embodiments, costimulation by one or more costimulatory moieties can be used to activate target cells, including T cells. In some embodiments, co-stimulation can be achieved by activating one or more cell surface markers, including but not limited to CD28, ICOS, CTLA4, PD1, PD1H, and BTLA. In some embodiments, the co-stimulatory moiety is an antibody.

[0248] Suitable payloads for the nanocapsules include HPRT-targeting synthetic oligonucleotides, shRNAs, miRNAs, and Ago-shRNAs. In some embodiments, payloads may be expressed with Pol III or Pol II driven promoter cassettes.

[0249] In other embodiments, agents for down-regulating HPRT may be formulated in bio-nanocapsules, which are nano-sized capsules produced by genetically engineered microorganisms. In some embodiments, the bio-nanocapsules are viral protein-derived or modified viral protein-derived particles, such as viral surface antigen particles (eg, hepatitis B virus surface antigen (HBsAg) particles). In other embodiments, the bio-nanocapsule is a nano-sized capsule comprising a lipid bilayer membrane and viral protein-derived or modified viral protein-derived particles, such as viral surface antigen particles. The particles can be purified from eukaryotic cells such as yeast, insect cells, and mammalian cells. Capsule sizes may range between about 10 nm and about 500 nm. In other embodiments, the capsule size may range between about 20 nm and about 250 nm. In still other embodiments, the capsule size may range between about 80 nm and about 150 nm.

[0250] antisense RNA Antisense RNA (asRNA) is a single-stranded RNA that is complementary to the intracellularly transcribed messenger RNA (mRNA) strand. While not wishing to be bound by any particular theory, it is believed that antisense RNAs are introduced into cells to inhibit the translation of complementary mRNAs by base-pairing with complementary mRNAs and physically interfering with the translational machinery. Stated another way, antisense RNA is a single-stranded RNA molecule that exhibits a complementary relationship with a specific mRNA.

[0251] Antisense RNA is utilized for gene regulation to specifically target mRNAs used for protein synthesis. Antisense RNAs physically pair and bind complementary mRNAs, thus inhibiting their ability to be processed in the translational machinery. In some embodiments, phosphorothioate-modified antisense oligonucleotides are utilized to target sequences within the coding region of HPRT mRNA. These oligonucleotides can be delivered to specific cell populations and anatomical sites using targeted nanoparticles, as described above.

[0252] exon skipping As described herein, exon skipping may be used to create a defect within the HPRT1 gene resulting in HPRT deficiency. In some embodiments, oligonucleotides (including modified oligonucleotides) may be delivered by nanocapsules, where the oligonucleotides are designed to target unspliced ​​HPRT mRNA and mediate premature termination or skipping of introns required for activity. Introduction of HPRT duplication mutations, such as duplication mutations in exon 4 (see Baba S, et al., "Novel mutation in HPRT1 causing a splicing error with multiple variations," Nucleosides Nucleotides Nucleic Acids. 2017 Jan. 2;36(1):1-6) could lead to splicing errors and functional inactivation of the HPRT protein. Replacing HPRT with an altered mutated sequence by spliceosomal trans-splicing is a potential therapeutic strategy for knocking down HPRT. This would require (1) a mutated coding region to replace the coding sequence in the target RNA, (2) a 5' or 3' splice site, and (3) a binding domain, e.g., an antisense oligonucleotide sequence, that is complementary to the target RNA.

[0253] An oligonucleotide may be structurally modified so that it is nuclease resistant. In some embodiments, oligonucleotides have modified backbones or non-natural internucleoside linkages. The oligonucleotides with modified backbones include those that retain the phosphorus atom in the backbone and those that lack the phosphorus atom in the backbone. In some embodiments, modified oligonucleotides that lack a phosphorus atom in their internucleoside backbone can also be considered oligonucleotides. In other embodiments, both the sugar and the internucleoside linkage, ie the backbone, of the nucleotide units are modified such that they are replaced with novel groups. Base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and attached directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also contain nucleobase (often referred to in the art simply as "base") modifications or substitutions. Certain nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds of the disclosure. These include, without limitation, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by about 0.6 to about 1.2°C, and are more particularly currently preferred base substitutions when combined with 2'-O-methoxyethyl sugar modifications.

[0254] Gene editing to knock out HPRT The disclosure also provides compositions for knocking out HPRT1. As a non-limiting example, isolated cells (e.g., primary T lymphocytes) can be treated with HPRT-targeted CRISPR / Cas, such as HPRT-targeted CRISPR / Cas9 RNP, HPRT-targeted CRISPR / Cas12a RNP, or HPRT-targeted CRISPR / Cas12b RNP.

[0255] A "ribonucleoprotein complex" as provided herein is a complex or particle comprising a nucleoprotein and a ribonucleic acid. A "nucleoprotein" as provided herein is a protein that can bind to nucleic acids (eg, RNA, DNA). When a nucleoprotein binds to ribonucleic acid, it is called a "ribonucleoprotein". Interactions between nucleoproteins and ribonucleic acids may be direct, e.g., covalent, or indirect, e.g., non-covalent (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole dipoles, dipole-induced dipoles, London dispersion), ring stacking (Pi effect), hydrophobic interactions, and the like).

[0256] In some embodiments, the ribonucleoprotein comprises an RNA binding motif non-covalently attached to the ribonucleic acid. For example, positively charged aromatic amino acid residues (eg, lysine residues) in RNA-binding motifs can form electrostatic interactions with the negative nucleic acid phosphate backbone of RNA, thereby forming ribonucleoprotein complexes. Non-limiting examples of ribonucleoproteins include ribosomes, telomerase, RNAseP, hnRNPs, CRISPR-associated protein 9 (Cas9) and small nuclear RNPs (snRNPs).

[0257] In some embodiments, the ribonucleoprotein can be an enzyme. In some embodiments the ribonucleoprotein is an endonuclease. Thus, in some embodiments, a ribonucleoprotein complex comprises an endonuclease and a ribonucleic acid. In some embodiments, the endonuclease is CRISPR-related protein 9. In some embodiments, the endonuclease is CRISPR-associated protein 12a. In some embodiments, the endonuclease is CRISPR-associated protein 12b.

[0258] In some embodiments, the ribonucleic acid is guide RNA. Examples of guide RNAs or guide RNA molecules include any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the guide RNA comprises one or more RNA molecules (eg, crRNA that is complementary to the target sequence and tracrRNA that functions as a binding scaffold for nucleases).

[0259] In some embodiments, the gRNA comprises a nucleotide sequence complementary to the target sequence (e.g., a target sequence within the X chromosome, a target sequence with the HPRT1 gene, a target sequence within exon 2, a target sequence within exon 3 of the HPRT1 gene, or a target sequence within exon 8 of the HPRT1 gene) or a portion thereof. A target sequence provided herein refers to a nucleic acid sequence that is expressed by a cell. In some embodiments, the target nucleic acid sequence is a foreign nucleic acid sequence. In some embodiments, the target sequence is an endogenous nucleic acid sequence. In some embodiments, the target sequence forms part of a cellular gene. Thus, in some embodiments the guide RNA is complementary to a cellular gene or fragment thereof.

[0260] In some embodiments, the guide RNA is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% complementary to the target nucleic acid sequence. In some embodiments, the guide RNA is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the sequence of the cellular gene. In some embodiments, the guide RNA binds to cellular gene sequences.

[0261] In some embodiments, the complementary strand of the guide RNA has at least about 50% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 55% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 60% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 65% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 70% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 75% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 80% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 85% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 90% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 95% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 96% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 97% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 98% sequence identity with the target sequence. In some embodiments, the complementary strand of the guide RNA has at least about 99% sequence identity with the target sequence. In some embodiments the guide RNA comprises a target sequence.

[0262] In some embodiments, the disclosure provides compositions comprising a guide RNA that targets a sequence (SEQ ID NO: 12) within the human hypoxanthine phosphoribosyltransferase (HPRT) gene. In some embodiments, the composition comprises a guide RNA that targets a sequence within the human X chromosome at a position in the range of about 134460145 to about 134500668 (based on GRCh38 or an equivalent position in a genome build other than GRCh38 (e.g., a previously known or future genome build)). In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 14 to about 28 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or an equivalent position in genome builds other than GRCh38), and the targeted sequence has a length ranging from about 15 to about 26 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or an equivalent position in genome builds other than GRCh38), and the targeted sequence has a length ranging from about 16 to about 24 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 17 to about 22 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 18 to about 22 contiguous base pairs. Locations within chromosome X are referenced herein to Genome Reference Consortium Human Build 38 (GRCh38), although those skilled in the art understand that these reference locations may have moved to equivalent locations in alternative human genome builds or assemblies.

[0263] In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 90% sequence identity with a target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 95% sequence identity with a target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 96% sequence identity to a target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 97% sequence identity with a target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 98% sequence identity to a target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 99% sequence identity to a target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38).

[0264] In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 90% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 91% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 92% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 93% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 94% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 95% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61.

[0265] In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 96% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 97% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 98% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61. In some embodiments, the complementary strand of the target sequence located at a position ranging from about 134460145 to about 134500668 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38) has at least 99% sequence identity with any one of SEQ ID NOs:25-39 or any one of SEQ ID NOs:40-61.

[0266] In some embodiments, the composition comprises a guide RNA that targets a sequence within the human X chromosome at a position ranging from about 134475181 to about 134475364 (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 14 to about 28 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 15 to about 26 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 16 to about 24 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 17 to about 24 contiguous base pairs. In some embodiments, the composition comprises a gRNA targeting a sequence having a position ranging from about 134475181 to about 134475364 within the X chromosome, and the targeted sequence has a length ranging from about 18 to about 24 contiguous base pairs.

[0267] In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 90% sequence identity to a target sequence located at a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 95% sequence identity to a target sequence located at a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 96% sequence identity to a target sequence located at a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 97% sequence identity to a target sequence located at a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 98% sequence identity to a target sequence located at a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 99% sequence identity to a target sequence located at a position ranging from about 134475181 to about 134475364 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38).

[0268] In some embodiments, the composition comprises a guide RNA that targets a sequence within the human X chromosome at a position ranging from about 134498608 to about 134498684 (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 14 to about 28 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 15 to about 26 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 16 to about 24 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 17 to about 24 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 18 to about 24 contiguous base pairs.

[0269] In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 90% sequence identity with a target sequence located at a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 95% sequence identity to a target sequence located at a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 96% sequence identity to a target sequence located at a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 97% sequence identity to a target sequence located at a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 98% sequence identity with a target sequence located at a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 99% sequence identity to a target sequence located at a position ranging from about 134498608 to about 134498684 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38).

[0270] In some embodiments, the guide RNA has at least 85% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the guide RNA has at least 85% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the guide RNA has at least 90% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the guide RNA has at least 97% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the guide RNA has at least 99% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the guide RNA has a nucleotide sequence comprising any one of SEQ ID NOs:25-39.

[0271] In some embodiments, the composition comprises a guide RNA that targets a sequence within the human X chromosome at a position ranging from about 134473409 to about 134473460 (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 14 to about 28 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 15 to about 26 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 16 to about 24 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 17 to about 24 contiguous base pairs. In some embodiments, the composition comprises a guide RNA targeting a sequence having a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or an equivalent position in a genome build other than GRCh38), and the targeted sequence has a length ranging from about 18 to about 24 contiguous base pairs.

[0272] In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 90% sequence identity to a target sequence located at a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 95% sequence identity to a target sequence located at a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 96% sequence identity with a target sequence located at a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 97% sequence identity to a target sequence located at a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 98% sequence identity to a target sequence located at a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38). In some embodiments, the composition comprises a gRNA having a nucleotide sequence that has at least 99% sequence identity to a target sequence located at a position ranging from about 134473409 to about 134473460 within the X chromosome (based on genome build GRCh38 or equivalent positions in genome builds other than GRCh38).

[0273] In some embodiments, the guide RNA has at least 85% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 85% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 90% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 91% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 92% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 93% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 94% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 96% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 97% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 98% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has at least 99% sequence identity with any one of SEQ ID NOs:40-56. In some embodiments, the guide RNA has a nucleotide sequence comprising any one of SEQ ID NOs:40-56.

[0274] In some embodiments, the guide RNA has at least 85% sequence identity with any one of SEQ ID NOs:57-61. In some embodiments, the guide RNA has at least 85% sequence identity with any one of SEQ ID NOs:57-61. In some embodiments, the guide RNA has at least 90% sequence identity with any one of SEQ ID NOs:57-61. In some embodiments, the guide RNA has at least 95% sequence identity with any one of SEQ ID NOs:57-61. In some embodiments, the guide RNA has at least 96% sequence identity with any one of SEQ ID NOs:57-61. In some embodiments, the guide RNA has at least 97% sequence identity with any one of SEQ ID NOs:57-61. In some embodiments, the guide RNA has at least 98% sequence identity with any one of SEQ ID NOs:57-61. In some embodiments, the guide RNA has at least 99% sequence identity with any one of SEQ ID NOs:57-61. In some embodiments, the guide RNA has a nucleotide sequence comprising any one of SEQ ID NOs:57-61.

[0275] In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 85% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 91% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 92% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 93% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 94% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 95% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 96% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 97% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 98% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 99% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having nucleotides comprising any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA having nucleotides comprising SEQ ID NO:25. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA having nucleotides comprising SEQ ID NO:26. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA having nucleotides comprising SEQ ID NO:27. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA having nucleotides comprising SEQ ID NO:28. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA having nucleotides comprising SEQ ID NO:29. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having nucleotides comprising SEQ ID NO:30. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having nucleotides comprising SEQ ID NO:31. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA having nucleotides comprising SEQ ID NO:32. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA having nucleotides comprising SEQ ID NO:33. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is guide RNA having nucleotides comprising SEQ ID NO:34. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having nucleotides comprising SEQ ID NO:35. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having nucleotides comprising SEQ ID NO:36. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having nucleotides comprising SEQ ID NO:37.

[0276] In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 85% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 91% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 92% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 93% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 94% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 95% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 96% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 97% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 98% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 99% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas9 and the ribonucleic acid is a guide RNA having nucleotides comprising any one of SEQ ID NOs:40-49.

[0277] In some embodiments, the endonuclease is Casl2a and the ribonucleic acid is guide RNA. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 85% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 91% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 92% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 93% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 94% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 95% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 96% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 97% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 98% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 99% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having nucleotides comprising any one of SEQ ID NOs:25-39.

[0278] In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 85% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 91% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 92% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 93% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 94% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 95% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 96% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 97% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 98% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 99% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having nucleotides comprising any one of SEQ ID NOs:40-49.

[0279] In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 85% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 91% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 92% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 93% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 94% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 95% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 96% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 97% sequence identity to any one of SEQ ID NOS:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 98% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 99% sequence identity to any one of SEQ ID NOs:50-61. In some embodiments, the endonuclease is Cas12a and the ribonucleic acid is a guide RNA having nucleotides comprising any one of SEQ ID NOs:40-49.

[0280] In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is guide RNA. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 85% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 91% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 92% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 93% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 94% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 95% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 96% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 97% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 98% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 99% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having nucleotides comprising any one of SEQ ID NOs:25-39.

[0281] In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 85% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 91% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 92% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 93% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 94% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 95% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 96% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 97% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 98% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having a nucleotide sequence with at least 99% sequence identity to any one of SEQ ID NOs:40-49. In some embodiments, the endonuclease is Cas12b and the ribonucleic acid is a guide RNA having nucleotides comprising any one of SEQ ID NOs:40-49.

[0282] host cell The disclosure also provides host cells containing the novel expression vectors of the disclosure. By "host cell" or "target cell" is meant a cell that is to be transformed (i.e., transduced or transfected) using a composition, e.g., an expression vector or nanocapsule, of the present disclosure. In some embodiments, the host cell becomes substantially HPRT deficient following transduction with an expression vector encoding a nucleic acid adapted to knockdown HPRT. In other embodiments, the host cell becomes substantially HPRT deficient following transfection with nanocapsules containing components designed to achieve knockout of HPRT. Methods for transducing host cells with expression vectors that knock down HPRT or transfecting host cells with nanocapsules that knock out HPRT are described in co-pending U.S. Patent Application No. 16 / 038,643, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, host cells are isolated and / or purified.

[0283] In some embodiments, host cells are mammalian cells capable of expressing expression vectors. Suitable mammalian host cells include, but are not limited to, human cells, mouse cells, non-human primate cells (e.g., rhesus monkey cells), human progenitor or stem cells, 293 cells, HeLa cells, D17 cells, MDCK cells, BHK cells, and Cf2Th cells. In certain embodiments, host cells comprising expression vectors of the present disclosure are hematopoietic cells such as hematopoietic progenitor / stem cells (e.g., CD34-positive hematopoietic progenitor / stem cells), monocytes, macrophages, peripheral blood mononuclear cells, CD4+ T lymphocytes, CD8+ T lymphocytes, or dendritic cells.

[0284] Hematopoietic cells (e.g., CD4+ T lymphocytes, CD8+ T lymphocytes and / or monocytes / macrophages) transduced with expression vectors of the present disclosure or transfected with nanocapsules can be of allogeneic, autologous, or matched sibling origin. Hematopoietic progenitor / stem cells, in some embodiments, are CD34 positive and can be isolated from the patient's bone marrow or peripheral blood. Isolated CD34-positive hematopoietic progenitor / stem cells (and / or other hematopoietic cells described herein) are, in some embodiments, transduced with an expression vector described herein.

[0285] In some embodiments, the modified host cells are combined with a pharmaceutically acceptable carrier. In some embodiments, host cells or transduced host cells are formulated in PLASMA-LYTE A (e.g., a sterile, non-pyrogenic, isotonic solution for intravenous administration; in which case 1 liter of PLASMA-LYTE A has ionic concentrations of 140 mEq sodium, 5 mEq potassium, 3 mEq magnesium, 98 mEq chloride, 27 mEq acetate, and 23 megluconate). In other embodiments, host cells or transduced host cells are formulated with a solution of PLASMA-LYTE A, a solution containing between about 8% and about 10% dimethylsulfoxide (DMSO). In some embodiments, about 2 x 10 7 Fewer than 10 host cells / transduced host cells are present per mL of formulation containing PLASMA-LYTE A and DMSO.

[0286] In some embodiments, the host cell becomes substantially HPRT deficient following transduction with an expression vector according to the present disclosure. In some embodiments, the level of HPRT1 gene expression is reduced by at least about 80%. Cells with 20% or less residual HPRT1 gene expression would be sensitive to purine analogues such as 6-TG or 6-MP, allowing their selection with purine analogues (see, eg, FIG. 22). In some embodiments, the host cell comprises a nucleic acid molecule having at least 90% identity to at least one of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the host cell comprises a nucleic acid molecule having at least 95% identity to at least one of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the host cell comprises a nucleic acid molecule comprising at least one of SEQ ID NO:3 or SEQ ID NO:4.

[0287] In some embodiments, transduction of host cells may be increased by contacting host cells in vitro, ex vivo, or in vivo with an expression vector of the present disclosure and one or more compounds that increase transduction efficiency. For example, in some embodiments, the one or more compounds that increase transduction efficiency are compounds that stimulate the prostaglandin EP receptor signaling pathway, i.e., one or more compounds that increase the downstream cell signaling activity of the prostaglandin EP receptor in cells contacted with the one or more compounds compared to the downstream cell signaling activity of the prostaglandin EP receptor in the absence of the one or more compounds. In some embodiments, the one or more compounds that increase transduction efficiency are prostaglandin EP receptor ligands, including, but not limited to, prostaglandin E2 (PGE2), or analogs or derivatives thereof. In other embodiments, the one or more compounds that increase transduction efficiency include, but are not limited to, Retronectin (63 kD fragment of recombinant human fibronectin fragment, available from Takara); Lentiboost (membrane sealing poloxamer, available from Sirion Biotech), protamine sulfate, cyclosporine H, and rapamycin. In still other embodiments, the one or more compounds that increase transduction efficiency comprise a poloxamer (eg, poloxamer F127).

[0288] In some embodiments, the host cell is prepared by contacting the host cell with a composition comprising a component that knocks out the HPRT1 gene from the host cell. In some embodiments, the HPRT1 gene knockout components comprise an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 85% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout components comprise an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 90% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout component comprises an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 91% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout component comprises an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 92% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout components comprise an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 93% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout components comprise an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 94% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout components comprise an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 95% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout components comprise an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 96% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout component comprises an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 97% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout component comprises an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 98% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout components comprise an endonuclease (e.g., Cas9, Cas12a, or Cas12b) and a guide RNA molecule having at least 99% sequence identity to any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61. In some embodiments, the HPRT1 gene knockout components comprise an endonuclease (eg, Cas9, Cas12a, or Cas12b) and a guide RNA molecule comprising any one of SEQ ID NOs:25-39 or SEQ ID NOs:40-61.

[0289] Pharmaceutical composition The disclosure also provides compositions, including pharmaceutical compositions, that include one or more of the expression vectors and / or non-viral delivery vehicles (eg, nanocapsules) disclosed herein. In some embodiments, pharmaceutical compositions comprise an effective amount of at least one of the expression vectors and / or non-viral delivery vehicles described herein and a pharmaceutically acceptable carrier. For example, in certain embodiments, a pharmaceutical composition comprises an effective amount of an expression vector and a pharmaceutically acceptable carrier. Effective amounts can be readily determined by those of ordinary skill in the art based on factors such as body size, weight, age, health, sex, ethnicity, and viral titer of the subject.

[0290] Another aspect of the disclosure is a pharmaceutical composition comprising (a) an expression vector comprising a nucleic acid sequence encoding an shRNA that targets the HPRT1 gene; and (b) a pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions are formulated as emulsions. In some embodiments, the pharmaceutical composition is formulated within micelles. In some embodiments, the pharmaceutical composition is encapsulated within the polymer. In some embodiments, pharmaceutical compositions are encapsulated within liposomes. In some embodiments, the pharmaceutical composition is encapsulated within minicells or nanocapsules.

[0291] In some embodiments, the pharmaceutical composition comprises (a) a population of nanocapsules, each nanocapsule comprising a payload (e.g., a Cas9 protein, a Cas12a protein, a Cas12b protein, and / or a gRNA such as the gRNA of any one of SEQ ID NOs: 25-39) to knockout HPRT adapted to it; and (b) a pharmaceutically acceptable carrier. In some embodiments, the nanocapsules are polymeric nanocapsules. In some embodiments, the polymeric nanocapsules further comprise at least one targeting moiety that facilitates delivery of the ribonucleoprotein or ribonucleoprotein complex to specific types of cells. In some embodiments, polymeric nanocapsules are erodible or biodegradable. In some embodiments, the polymeric nanocapsules comprise pH sensitive crosslinkers. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127 or FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0292] In some embodiments, the pharmaceutical composition comprises (a) a population of nanocapsules, each nanocapsule having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity to any one of matched knockout HPRTs (e.g., Cas9 protein, Cas12a protein, Cas12b protein and / or gRNA, e.g., SEQ ID NOs: 40-61 , at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity), and (b) a pharmaceutically acceptable carrier. In some embodiments, the nanocapsules are polymeric nanocapsules. In some embodiments, the polymeric nanocapsules further comprise at least one targeting moiety that facilitates delivery of the ribonucleoprotein or ribonucleoprotein complex to specific types of cells. In some embodiments, polymeric nanocapsules are erodible or biodegradable. In some embodiments, the polymeric nanocapsules comprise pH sensitive crosslinkers. In some embodiments, at least one targeting moiety targets a T cell marker. In some embodiments, the T cell marker is selected from CD3, CD4, CD7, CD8, CD25, CD27, CD28, CD45RA, RO, CD56, CD62L, CD127 or FoxP3 and CD44. In some embodiments the T cell marker is CD3. In some embodiments, the T cell marker is CD28.

[0293] In some embodiments, polymeric nanocapsules have sizes ranging between about 50 nm and about 250 nm. In some embodiments, the polymeric nanocapsules have an average diameter less than or equal to about 200 nanometers (nm). In some embodiments, polymeric nanocapsules have an average diameter between about 1-200 nm. In some embodiments, polymeric nanocapsules have an average diameter between about 5 and about 200 nm. In some embodiments, polymeric nanocapsules have an average diameter between about 10 and about 150 nm, or between 15 and 100 nm. In some embodiments, polymeric nanocapsules have an average diameter between about 15 and about 150 nm. In some embodiments, polymeric nanocapsules have an average diameter between about 20 and about 125 nm. In some embodiments, polymeric nanocapsules have an average diameter between about 50 and about 100 nm. In some embodiments, polymeric nanocapsules have an average diameter between about 50 and about 75 nm. In some embodiments, the surface of the nanocapsules can have a charge (eg, measured in standard phosphate solution) between about 1 and about 15 millivolts (mV). In other embodiments, the surface of the nanocapsules can have a charge between about 1 and about 10 mV.

[0294] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. For example, an expression vector is formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like suitable for use in formulating pharmaceuticals such as pharmaceuticals suitable for administration to humans. Methods for formulating compounds with pharmaceutical carriers are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, 17th ed. Mack Publishing Company, Easton, Pa. 1985; and Goodman & Gillman's: The Pharmacological Basis of Therapeutics (11th ed., McGraw-Hill Professional, 2005); incorporate into the book.

[0295] In some embodiments, a pharmaceutical composition may comprise any expression vector, nanocapsule, or composition disclosed herein at any concentration that enables the administered silencing nucleic acid to achieve a concentration ranging from about 0.1 mg / kg to about 1 mg / kg. In some embodiments, the pharmaceutical composition may contain the expression vector in an amount of about 0.1% to about 99.9% of the total weight of the pharmaceutical composition. In some embodiments, the pharmaceutical composition may contain the expression vector in an amount of about 0.1% to about 90% by weight of the total pharmaceutical composition. Pharmaceutically acceptable carriers suitable for inclusion within any pharmaceutical composition include water, buffered water, e.g., saline such as normal saline or balanced saline such as Hank's or Earle's balanced solutions, glycine, hyaluronic acid, and the like. Pharmaceutical compositions may be formulated for parenteral administration such as intravenous, intramuscular or subcutaneous administration. Pharmaceutical compositions for parenteral administration may include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersion. Examples of suitable water-soluble and non-aqueous carriers, solvents, diluents or vehicles include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and mixtures thereof, vegetable oils (such as olive oil), injectable organic esters (such as ethyl oleate).

[0296] Pharmaceutical compositions are formulated for oral administration. Solid dosage forms for oral administration may include, for example, tablets, dragees, capsules, pills, and granules. In such solid dosage forms, the composition comprises at least one pharmaceutically acceptable carrier such as sodium citrate and / or dicalcium phosphate and / or fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; binders such as carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose and acacia; humectants such as glycerin; agar, calcium carbonate, potato or tapioca starch, alginic acid, silicates, and Wetting agents such as acetyl alcohol, glycerin monostearate; absorbants such as kaolin and bentonite clays; and / or lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. Liquid dosage forms for oral administration may include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. Liquid formulations include inert diluents such as water or other solvents, solubilizers and / or emulsions such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil, corn oil, germ oil, castor oil, olive oil, sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. good too.

[0297] Pharmaceutical compositions may include penetration enhancers to enhance their delivery. Penetration enhancers may include fatty acids such as oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linoleic acid, dicaprate, reclineate, monoolein, dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, mono- and di-glycerides and physiologically acceptable salts thereof. The composition may further comprise a chelating agent such as, for example, ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (eg, sodium salicylate, 5-methoxysalicylate, homovanillate).

[0298] A pharmaceutical composition may include any of the expression vectors disclosed herein in encapsulated form. For example, the expression vector is encapsulated by biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides), or encapsulated in liposomes or dispersed in microemulsions. Liposomes can be, for example, lipofectin or lipofectamine. In another example, a composition may comprise an expression vector disclosed herein in or on an anuclear bacterial minicell (Giacalone et al., Cell Microbiology 2006, 8(10):1624-33). The expression vectors disclosed herein may be combined with nanoparticles.

[0299] Stable production cell line Another aspect of the present disclosure is a stable producer cell line for generating viral titers, the stable producer cell line being derived from one of the GPR, GPRG, GPRT, GPRGT, or GPRT-G packing cell lines. In some embodiments, the stable producer cell line is derived from the GPRT-G cell line. In some embodiments, the stable production cell line (a) synthesizes an expression vector by cloning a nucleic acid sequence encoding at least an anti-HPRT shRNA into a recombinant plasmid (i.e., the synthesized vector can be any one of the vectors described herein); (b) generates DNA fragments from the synthesized vector; (c) forms a concatemer array from (i) DNA fragments generated from the synthesized vector, and (ii) DNA fragments from an antibiotic resistance cassette plasmid; generated by transfecting the formed concatemer array into one of the cell lines; and (e) isolating a stable producer cell line. Additional methods of forming stable production cell lines are disclosed in International Application PCT / US2016 / 031959 filed May 12, 2016, the disclosure of said patent document is hereby incorporated herein by reference in its entirety.

[0300] kit Some embodiments include kits that include the expression vectors or compositions that include the expression vectors described herein. A kit may include a container, which may be a bottle containing the expression vector or composition in oral or parenteral dosage form, each dosage form containing a unit dose of the expression vector. Kits may include a label or the like that directs treatment of a subject according to the methods described herein. Similarly, in another embodiment is a kit comprising a composition comprising a population of nanocapsules comprising a payload adapted to knockout HPRT as described herein.

[0301] In some embodiments, the kit may contain additional active agents. The additional active agent may be contained in a container separate from the container containing the vector or vector-containing composition. For example, in some embodiments, a kit may include one or more doses of a purine analogue (e.g., 6-TG or 6-MP) and optionally instructions for administering the purine analogue for pretreatment and / or chemoselection (the steps for which are further described herein). In other embodiments, the kit may include one or more doses of MTX or MPA and, optionally, instructions for administering MTX or MPA for negative selection as described herein.

[0302] Production of substantially HPRT-deficient lymphocytes (“altered lymphocytes”) One aspect of the present disclosure is a method of producing HPRT-deficient lymphocytes, eg, T cells (also referred to herein as "altered lymphocytes" or "altered T cells"). With reference to Figure 11, host cells, ie lymphocytes (eg, T cells), are first harvested from a donor (step 110). In embodiments in which hematopoietic stem cells (HSCs) are also collected from a donor, lymphocytes, eg, T cells, may be collected from the same donor from which the HSC graft was collected or from a different donor. In these embodiments, the cells may be harvested at the same time as the cells for the HSC graft or at different times. In some embodiments, cells are harvested from the same mobilized peripheral blood HSC harvest. In some embodiments, this could be the CD34-negative fraction (CD34-positive cells harvested by standard treatment for donor grafts) or a portion of the CD34-positive HSC graft if a precursor T-cell graft is envisaged.

[0303] Those skilled in the art will recognize that cells may be collected by any means. For example, cells may be collected through apheresis, leukoapheresis, or simply simple venous bleeding. In embodiments in which the HSC graft is harvested at the same time as the cells for modification, the HSC graft is cryopreserved to allow time for manipulation and testing of the harvested lymphocytes, eg, T cells. Non-limiting examples of T cells include T helper T cells (eg, Th1, Th2, Th9, Th17, Th22, Tfh), regulatory T cells, natural killer T cells, gamma delta T cells, and cytotoxic lymphocytes (CTL).

[0304] Following cell harvesting, lymphocytes, eg, T cells, are isolated (step 120). Lymphocytes, eg, T cells, may be isolated from collected cell aggregates by any means known to those of skill in the art. For example, CD3+ cells may be isolated from collected cells by CD3 microbeads and MACS separation system (Miltenyi Biotec). The CD3 marker is expressed on all T cells and is believed to be associated with the T cell receptor. About 70-80% of human peripheral blood lymphocytes and about 65-85% of thymocytes are believed to be CD3+. In some embodiments, the CD3+ cells are magnetically labeled with CD3 microbeads. The cell suspension is then loaded onto a MACS column placed in the magnetic field of a MACS separator. Magnetically labeled CD3+ cells are retained on the column. Unlabeled cells flow through and this cell fraction is depleted of CD3+ cells. After removing the column from the magnetic field, the magnetically retained CD3+ cells can be eluted as the positively selected cell fraction.

[0305] Alternatively, CD62L+ T cells are isolated from harvested cells by the IBA life sciences CD62L Fab Streptamer isolation kit. Isolation of human CD62L+ T cells is performed by positive selection. PBMC are labeled with magnetic CD62L Fab Streptamers. Labeled cells are isolated on a strong magnet where they migrate towards the tube wall on the magnet side. This CD62L positive cell fraction is collected and the cells are released from all labeling reagents by adding biotin to a strong magnet. Magnetic Streptamers migrate toward the tube wall, leaving unlabeled cells in the supernatant. Biotin is removed by washing. The resulting cell preparation is highly enriched for CD62L+ T cells with >90% purity. No depletion step or column is required.

[0306] In an alternative embodiment, lymphocytes, eg, T cells, are not isolated in step 120, but rather aggregates of cells collected in step 110 are used for subsequent modification. In some embodiments, aggregates of cells may be used for subsequent modification, although in some instances the method of modification may be specific to a particular cell population within the overall aggregate of cells. This could be accomplished in a number of ways, e.g., by targeted gene vector delivery, or by directing the genetic modification to specific cell types, e.g., by directing the expression of the shRNA against HPRT to specific cell types, i.e., T cells.

[0307] Following isolation of the T cells, the T cells are treated to reduce HPRT activity (step 130), ie, reduce expression of the HPRT1 gene. For example, T cells can be treated with about 50% or less residual HPRT1 gene expression, about 45% or less residual HPRT1 gene expression, about 40% or less residual HPRT1 gene expression, about 35% or less residual HPRT1 gene expression, about 30% or less residual HPRT1 gene expression, about 25% or less residual HPRT1 gene expression, about 20% or less residual HPRT1 gene expression. It may be treated to have gene expression, about 15% or less of residual HPRT1 gene expression, about 10% or less of residual HPRT1 gene expression, or about 5% or less of residual HPRT1 gene expression.

[0308] Lymphocytes, such as T cells, are modified according to several methods. In some embodiments, T cells may be modified by transduction with an expression vector, eg, a lentiviral vector, encoding an shRNA directed against the HPRT1 gene as described herein. For example, the expression vector can include a first expression control sequence operably linked to a first nucleic acid sequence encoding an shRNA that knocks down HPRT and has at least 90% identity to the sequence of any of SEQ ID NOS: 2, 5, 6, and 7. As another example, the expression vector may comprise a first expression control sequence operably linked to a first nucleic acid sequence encoding an shRNA that knocks down HPRT and has at least 90% identity to the sequence of any of SEQ ID NOs:8, 9, 10, and 11. In some embodiments, the expression vector is encapsulated within a nanocapsule.

[0309] In some embodiments, an expression vector may include a first nucleic acid sequence encoding an endonuclease and a second nucleic acid sequence encoding a guide RNA. In some embodiments, the first nucleic acid sequence encodes Cas9. In some embodiments, the first nucleic acid sequence encodes Casl2a. In some embodiments, the first nucleic acid sequence encodes Casl2b. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 90% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 91% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 92% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 93% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 94% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 95% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 96% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 97% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 98% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 99% sequence identity with any one of SEQ ID NOs:25-39. In some embodiments, the second nucleic acid sequence encodes a guide RNA having any one of SEQ ID NOs:25-39.

[0310] In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 90% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 91% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 92% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 93% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 94% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 95% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 96% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 97% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 98% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having at least 99% sequence identity with any one of SEQ ID NOs:40-61. In some embodiments, the second nucleic acid sequence encodes a guide RNA having any one of SEQ ID NOs:40-61.

[0311] In some embodiments, lymphocytes, eg, T cells, may be modified by transfection with an endonuclease and guide RNA. In some embodiments, lymphocytes, eg, T cells, may be modified by transfection with particles comprising an endonuclease and guide RNA. In some embodiments, lymphocytes, e.g., T cells, may be modified by transfection with nanocapsules containing payloads adapted to knock out HPRT, i.e., HPRT may be knocked out using gene editing techniques. In some embodiments, lymphocytes, e.g., T cells, may be modified by transfection with targeted nanocapsules containing payloads adapted to knock out HPRT, i.e., HPRT may be knocked out using gene editing techniques.

[0312] For example, T cells may be treated with HPRT-targeted CRISPR / Cas9 RNP, CRISPR / Cas12a RNP, or CRISPR / Cas12b RNP as described herein. In some embodiments, the nanocapsules may contain guide RNAs having at least 90% sequence identity with any one of SEQ ID NOs:25-39. In other embodiments, the nanocapsules may contain guide RNAs with at least 95% sequence identity to any one of SEQ ID NOs:25-39. In some embodiments, the nanocapsules may contain guide RNAs having at least 90% sequence identity with any one of SEQ ID NOs:40-61. In other embodiments, the nanocapsule may contain guide RNAs having at least 95% sequence identity with any one of SEQ ID NOs:40-61.

[0313] After the T cells are modified in step 130, a population of HPRT-deficient T cells is selected and / or expanded (step 140). In some embodiments, the culture simultaneously selects and expands cells with enhanced engraftment capacity (eg, central memory or T stem cell phenotype). In some embodiments, the culture period is less than 14 days. In some embodiments, the culture period is less than 7 days.

[0314] In some embodiments, the step of picking out and expanding cells comprises treating a population of HPRT-deficient (or substantially HPRT-deficient) lymphocytes, e.g., T cells, with a guanosine analog antimetabolite (such as 6-thioguanine (6-TG), 6-mercaptopurine (6-MP), or azathioprine (AZA)) ex vivo. In some embodiments, lymphocytes, eg, T cells, are cultured in the presence of 6-thioguanine (“6-TG”), thus killing cells that were not modified in step 130. 6-TG is a guanine analogue that can interfere with dGTP biosynthesis in cells. Thio-dG can be incorporated into DNA in place of guanine during replication and is often methylated upon incorporation. This methylation can interfere with proper mismatch DNA repair and can lead to cell cycle arrest and / or initiate apoptosis. 6-TG has been used clinically to treat patients with certain types of malignancies because of its toxicity to rapidly dividing cells. In the presence of 6-TG, HPRT is the enzyme responsible for the incorporation of 6-TG into DNA and RNA in cells, blocking proper polynucleotide synthesis and metabolism (see Figure 18). On the other hand, the salvage pathway is blocked in HPRT-deficient cells (see Figure 18). Therefore, cells use a novel pathway for purine synthesis (see Figure 17). However, in HPRT wild-type cells, cells use the salvage pathway and 6-TG is converted to 6-TGMP in the presence of HPRT. 6-TGMP is converted to thioguanine diphosphate (TGDP) and thioguanine triphosphate (TGTP) by phosphorylation. At the same time, a deoxyribosyl analogue is formed by the enzyme ribonucleotide reductase. Given that 6-TG is highly cytotoxic, 6-TG can be used as a selective agent to kill cells together with functional HPRT enzymes.

[0315] The generated HPRT-deficient cells are then contacted ex vivo with a purine analogue. In the knockdown approach, there may still be residual HPRT in the cells, and HPRT-knockdown cells could tolerate a range of purine analogues, but would be killed at high doses / volumes. In this context, concentrations of purine analogues used for ex vivo selection range from about 15 μM to about 200 nM. In some embodiments, concentrations of purine analogs used for ex vivo selection range from about 10 μM to about 50 nM. In some embodiments, concentrations of purine analogs used for ex vivo selection range from about 5 μM to about 50 nM. In some embodiments, concentrations range from about 2.5 μM to about 10 nM. In other embodiments, concentrations range from about 2 μM to about 5 nM. In still other embodiments, concentrations range from about 1 μM to about 1 nM.

[0316] In the knockout approach, HPRT may be completely or almost completely removed from HPRT knockout cells, and the HPRT-deficient cells generated will be highly permissive to purine analogs. In some embodiments, concentrations of purine analogs used for ex vivo selection range from about 200 μM to about 5 nM in this case. In some embodiments, concentrations of purine analogs used for ex vivo selection range from about 100 μM to about 20 nM in this case. In some embodiments, concentrations range from about 80 μM to about 10 nM. In other embodiments, concentrations range from about 60 μM to about 10 nM. In still other embodiments, concentrations range from about 40 μM to about 20 nM.

[0317] In other embodiments, cell modification (e.g., through knockdown or knockout of HPRT) may be sufficiently efficient that ex vivo selection for HPRT-deficient cells is not required, i.e., selection with 6-TG or other similar compounds.

[0318] In some embodiments, the generated HPRT-deficient cells are contacted with both a purine analogue and with allopurinol, an inhibitor of xanthine oxidase (XO). By inhibiting XO, more available 6-TG will be metabolized by HPRT. When 6-TG is metabolized by HPRT, 6-TG forms 6-TGN, a metabolite that is toxic to cells (6-TGN includes monophosphate (6-TGMP), diphosphate (6-TGDP) and triphosphate (6-TGTP)) (see Figure 14). (For example, Curkovic et al., Low allopurinol doses are sufficient to optimize azathioprine therapy in inflammatory bowel disease patients with inadequate thiopurine metabolite concentrations. Eur J Clin Pharmacol. 2013 Aug;69(8):1521-31; Gardiner et al., Allopurinol might improve response to azathioprine and 6-mercaptopurine by correcting an unfavorable metabolite ratio. Gastroenterol Hepatol. 2011 Jan;26(1):49-54; Seinen et al., The effect of allopurinol and low-dose thiopurine combination therapy on the activity of three pivotal thiopurine metabolizing enzymes: results from a prospective pharmacological study. J Crohns Colitis. 2013 Nov;7(10):812-9; and Wall et al., Addition of Allopurinol for Alter. See ing Thiopurine Metabolism to Optimize Therapy in Patients with Inflammatory Bowel Disease.

[0319] In some embodiments, allopurinol is introduced into HPRT-deficient cells generated prior to introduction of the purine analogue. In other embodiments, allopurinol is introduced into HPRT-deficient cells generated concurrently with the introduction of the purine analogue. In still other embodiments, allopurinol is introduced into HPRT-deficient cells generated following introduction of the purine analogue.

[0320] Following selection and expansion, the modified lymphocytes, eg T cell products, are tested. In some embodiments, the modified lymphocytes, e.g., T cell products, are tested according to standard release tests (e.g., activity, mycoplasma, viability, stability, phenotype, etc.; see Molecular Therapy: Methods & Clinical Development vol.

[0321] In other embodiments, modified lymphocyte, eg, T cell products, are tested for sensitivity to dihydrofolate reductase inhibitors (eg, MTX or MPA). Dihydrofolate reductase inhibitors, including both MTX and MPA, inhibit de novo synthesis of purines, but are believed to have different mechanisms of action. For example, MTX is believed to competitively inhibit dihydrofolate reductase (DHFR), an enzyme involved in tetrahydrofolate (THF) synthesis. DHFR catalyzes the conversion of dihydrofolate to active tetrahydrofolate. Folic acid is required for de novo synthesis of the nucleoside thymidine required for DNA synthesis. Moreover, folate is essential for purine and pyrimidine base biosynthesis, so synthesis will be inhibited. Mycophenolic acid (MPA) is a potent, reversible, noncompetitive inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH), an enzyme essential for the de novo synthesis of guanosine-5'-monophosphate (GMP) from inosine-5'-monophosphate (IMP).

[0322] Thus, dihydrofolate reductase inhibitors, including both MTX or MPA, inhibit DNA, RNA, thymidylate, and protein synthesis. MTX or MPA block the de novo pathway by inhibiting ...

Claims

1. 1. A pharmaceutical composition comprising a population of modified lymphocytes, the population of modified lymphocytes comprising: (a) Lymphocytes obtained from a donor sample, (i) an endonuclease, and (ii) a guide RNA molecule that targets a sequence contained in one of exon 2, exon 3, or exon 8 of the HPRT1 gene. to generate a population of lymphocytes substantially deficient in HPRT; and (b) positively selecting ex vivo a population of lymphocytes that are substantially HPRT deficient to provide a population of modified lymphocytes. Generated by The pharmaceutical composition is administered by administering to the patient a therapeutically effective amount of the population of modified lymphocytes.

2. 10. The pharmaceutical composition of claim 1, further comprising the step of administering an HSC graft prior to, concurrently with, or after administration of the population of modified lymphocytes.

3. The pharmaceutical composition of claim 1, wherein the guide RNA molecule targeting exon 2 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs: 45 and 57-61.

4. The pharmaceutical composition of claim 1, wherein the guide RNA molecule targeting exon 2 of the HPRT1 gene comprises any one of SEQ ID NOs: 45 and 57-61.

5. The pharmaceutical composition of claim 1, wherein the guide RNA molecule targeting a sequence contained in one of exons 3 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs: 41-44, 46 and 50-51.

6. The pharmaceutical composition of claim 1, wherein the guide RNA molecule targeting a sequence contained in one of exons 3 of the HPRT1 gene comprises any one of SEQ ID NOs: 41-44, 46 and 50-51.

7. The pharmaceutical composition of claim 1, wherein the guide RNA molecule targeting a sequence contained in one of exons 8 of the HPRT1 gene has at least 95% sequence identity with any one of SEQ ID NOs: 47-49, 46, 55 and 56.

8. The pharmaceutical composition of claim 1, wherein the guide RNA molecule targeting a sequence contained in one of exons 8 of the HPRT1 gene comprises any one of SEQ ID NOs: 47-49, 46, 55 and 56.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the endonuclease comprises a Cas protein.

10. The pharmaceutical composition of claim 9, wherein the Cas protein comprises a Cas9 protein or a Cas12a protein.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein lymphocytes obtained from a donor sample are transfected or transduced by a viral delivery vehicle, a non-viral delivery vehicle, or a physical method.

12. The pharmaceutical composition of claim 11, wherein the physical method is selected from microinjection and electroporation.

13. The pharmaceutical composition of claim 11 , wherein the non-viral delivery vehicle is a nanocapsule, and optionally, the nanocapsule comprises at least one targeting moiety.

14. The pharmaceutical composition of claim 11, wherein the viral delivery vehicle is an expression vector, wherein the expression vector comprises a first nucleic acid sequence encoding an endonuclease and a second nucleic acid encoding a guide RNA molecule.

15. The pharmaceutical composition of claim 14 , wherein the expression vector is a lentiviral expression vector.

16. 16. The pharmaceutical composition of any one of claims 1 to 15, wherein the level of HPRT1 gene expression in the population of substantially HPRT-deficient lymphocytes is reduced by at least about 70%, preferably by at least about 80%, more preferably by at least about 90% compared to non-transfected donor lymphocytes.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the positive selection comprises contacting the resulting population of substantially HPRT-deficient lymphocytes with a purine analogue, preferably the purine analogue is selected from the group consisting of 6-TG and 6-MP.

18. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein positive selection comprises contacting the resulting population of substantially HPRT-deficient lymphocytes with both a purine analogue and allopurinol.

19. A pharmaceutical composition according to any one of claims 1 to 18, further comprising the step of administering to the patient one or more doses of a dihydrofolate reductase inhibitor if side effects occur.

20. 20. The pharmaceutical composition of claim 19, wherein the side effect is graft-versus-host disease (GvHD).

21. 21. The pharmaceutical composition of claim 19 or 20, wherein the dihydrofolate reductase inhibitor is selected from the group consisting of MTX or MPA.

22. 1. A modified lymphocyte preparation for providing the benefits of lymphocyte infusion to a subject in need of such treatment, the modified lymphocyte preparation comprising: (a) isolating lymphocytes from a donor subject; (b) separating the isolated lymphocytes from (i) an endonuclease, and (ii) a guide RNA molecule that targets a sequence contained in one of exon 2, exon 3, or exon 8 of the HPRT1 gene. to provide a population of lymphocytes that are substantially deficient in HPRT; and (c) exposing the population of HPRT-deficient lymphocytes to an agent that positively selects for HPRT-deficient lymphocytes, thereby providing a preparation of modified lymphocytes. A preparation produced by.

23. 13. A kit comprising: (i) a guide RNA molecule having at least 95% sequence identity to any one of SEQ ID NOs: 40-61; and (ii) Cas protein Kit including:

24. 24. The kit of claim 23, wherein the Cas protein is selected from the group consisting of a Cas9 protein and a Cas12 protein.