Mutagenesis to modify t cell functionality

Abstract

Provided herein are methods and compositions designed to enhance T cell functionality through precise base editing of genes within the RNA Pol II pre-initiation complex (PIC) and Mediator complex. The approach employs single guide RNAs (sgRNAs) targeting gene coding regions of the PIC or Mediator complexes alongside base editors, such as adenine or cytosine base editors, to facilitate targeted mutagenesis at the amino acid level. This process contributes to improved T cell attributes, including proliferation, cytokine production, IL2RA expression, and tumor infiltration. High-throughput screens are utilized to identify functional hotspots for therapeutic applications in cancer and autoimmune diseases. The method mitigates risks, such as double-strand breaks, commonly associated with traditional gene editing techniques and provides a platform for identifying therapeutic targets and drugs. Enhanced T cells generated through this methodology can be employed in cell therapy to strengthen immune responses.

Description

[0001] MUTAGENESIS TO MODIFY T CELL FUNCTIONALITY

[0002] PRIORITY

[0003] This application claims the benefit of the filing date of U.S. provisional application serial number 63 / 754,505, filed on February 5, 2025, and U.S. provisional application serial number 63 / 764,559, field on February 27, 2025, the disclosures of which are incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0005] This invention was made with government support under DK129364, and HG008140 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] FIELD OF THE INVENTION

[0007] The present disclosure relates to biotechnology, specifically to methods and compositions for enhancing T cell function through base editing of genes within the RNA Pol II pre-initiation complex and Mediator complex for therapeutic applications.

[0008] BACKGROUND

[0009] In recent years, T cell-based therapies have shown promise in oncology and autoimmunity by harnessing the body’s own immune system to target pathological cells. Existing approaches to T cell engineering have largely focused on complete gene disruption or ectopic gene expression, which often result in broad effects that can attenuate multiple pathways simultaneously. Such strategies may compromise viability, induce unintended immunogenicity, or fail to isolate sub-functional domains within multifunctional proteins. Moreover, traditional double-strand break-based gene editing can provoke p53-mediated responses, chromosomal rearrangements, and variable editing efficiencies across genomic loci.

[0010] SUMMARY

[0011] Provided herein is a method comprising contacting T cells with one or more sgRNAs provided in FIG. 15 targeted to one or more coding regions in one or more genes of the PIC or Mediator Complex and a base editor or nucleic acid encoding the base editor; and selecting one or more T cells that have enhance T cell function and optionally sorting T cells based on the profile.

[0012] In one embodiment, a method for engineering T cells includes contacting T cells with single-guide RNAs targeting coding regions of one or more genes encoding PIC or Mediator Complex subunits, together with a base editor or a nucleic acid encoding the base editor. The base editor can be an adenine base editor or a cytosine base editor. The sgRNAs may beprovided as a library expressed by viral vectors — such as lentivirus, retrovirus, adenovirus, herpesvirus or adeno-associated virus — and can reside on the same or on separate nucleic acid molecules from the base editor. Following editing, T cells exhibiting enhanced proliferation, increased IL2RA expression, altered cytokine production, greater tumor infiltration or modified IFN-y production, or exhibiting suppressed immune function, arc selected or sorted. The sgRNAs can comprise sequences from Figure 14 or SEQ ID NOs 1-20 (or sequences with at least 85% identity thereto), and the targeted coding regions can correspond to genes or sites shown in Figures 10, 14-16 (or sequences with at least 85% identity thereto).

[0013] In another embodiment, a cell population or an isolated human cell comprises a baseedited gene corresponding to one or more PIC or Mediator Complex subunit genes illustrated in Figures 14-16. The gene modification alters one or more cellular activities — such as enhanced T cell proliferation, increased IL2RA expression or cytokine production — relative to an unedited counterpart. The edited gene can be selected from MED24, MED16, TBP, GTF2A1, TAF3, ERCC3, GTF2F1, GTF2F2, CCNC, POLR2A, TAF9 or TAF8, with specific edits as illustrated in Figure 10.

[0014] In yet another embodiment, a method of preventing, inhibiting or treating cancer in a subject comprises administering a composition containing a plurality of the engineered cells. The cells can include CAR-T cells, be autologous or allogeneic, and are systemically administered to a human patient suffering from an immune cell cancer such as sarcoma, lymphoma, melanoma, carcinoma, lung cancer, breast cancer, prostate cancer, pancreatic cancer or ovarian cancer.

[0015] In a further embodiment, a method of preventing, inhibiting or treating an autoimmune disease comprises administering the engineered cells to a human subject in need thereof. The cells can be autologous or allogeneic, systemically administered, and are particularly suited for treatment of multiple sclerosis.

[0016] These and other features of the disclosure will be more fully understood from the following detailed description and accompanying claims.

[0017] DRAWINGS

[0018] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein.

[0019] FIG. 1. Results of IFNy in vivo screen using library of RNA Pol II pre -initiation complex general factors. Effect size of each donor represented as the log2FoldChange sgRNA (IFNy high bin / IFNy low bin). Edits are labeled as the gene name and predicted amino acid change.FIG. 2. Results of IFNy in vivo screen using library of Mediator complex general factors. Effect size of each donor represented as the log2FoldChange sgRNA (IFNy high bin / IFNy low bin). Edits are labeled as the gene name and predicted amino acid change.

[0020] FIG. 3. Results of in vivo tumor screen using library of RNA Pol II pre -initiation complex general factors. Effect size of each donor represented as the log2FoldChangc sgRNA (Tumor / Input). Edits are labeled as the gene name and predicted amino acid change.

[0021] FIG. 4. Results of in vitro stimulated CD4+ T cell IL2RA screen. Effect size of each donor represented as the log2FoldChange sgRNA (IL2RA high bin / IL2RA low bin). Edits are labeled as the gene name and predicted amino acid change.

[0022] FIG.5. Results of in vitro resting CD4+ T cell IL2RA screen. Effect size of each donor represented as the log2FoldChange sgRNA (IL2RA high bin / IL2RA low bin). Edits are labeled as the gene name and predicted amino acid change.

[0023] FIG. 6. Results of arrayed validation of IL2RA expression in human CD4+ T cells using flow cytometry. Each bar represents a guide and the time following stimulation is displayed on the y axis. The predicted edits are shown on the x axis. Log2Fold Change is calculated relative to the AAVS1 edit controls.

[0024] FIG. 7. Arrayed validation of IL2RA screen hits shows a strong positive correlation between surface protein expression (MFI) and screen Log2Fold Change.

[0025] FIGS. 8A-8C. A. Results of in vitro stimulated CD4+ T cell nascent RNA screen (Stim EU Screen) compared to Abundance screen. Effect size represented as the mean log2FoldChange sgRNA (EU high bin / EU low bin). Edits are labeled as the gene name and predicted amino acid change. The amino acid numbering in this figure is shifted by 1 relative to the provided tables (e.g., Figures 14-16 and 10) in the document so that the first amino acid is Ml V. B. Representation of select EU screen hits on published protein structure highlighting that disruption of CCNC increases nascent RNA production. The amino acid numbering in this figure is shifted by 1 relative to the provided tables (e.g., Figures 14-16 and 10) in the document so that the first amino acid is M1V. C. Validation of nascent RNA screen hits using flow cytometry wherein mutations in CCNC increase nascent RNA production in stimulated CD4+ T cells. The amino acid numbering in this figure is shifted by 1 relative to the provided tables (e.g., Figures 14-16 and 10) in the document so that the first amino acid is M1V.

[0026] FIGS. 9A-9C. A. Results of in vitro stimulated CD4+ T cell IL2RA screen vs abundance screen. Effect size represented as the mean log2FoldChange sgRNA (IL2RA high bin / IL2RA low bin). Edits are labeled as the gene name and predicted amino acid change. The amino acid numbering in this figure is shifted by 1 relative to the provided tables (e.g., Figures 14-16 and10) in the document so that the first amino acid is Ml V. B. Representation of select stimulated IL2RA screen hits on published protein structure highlighting that disruption of external residues on GTF2A1 decreases IL2RA expression. Notably, these residues do not decrease cell abundance as indicated in panel A. The amino acid numbering in this figure is shifted by 1 relative to the provided tables (c.g., Figures 14-16 and 10) in the document so that the first amino acid is Ml V. C. Depiction of select residues on MED24 and MED 16 that increase IL2RA expression on CD4+ T cells.

[0027] FIG. 10. Focused table of several sgRNAs and mutations.

[0028] FIGS. 11A-11C. POL2A_5093 base-edited CD 19 CAR T cells enhance melanoma tumor control in vivo and exhibit increased CAR T cell infiltration in both tumors and peripheral blood. A) Tumor growth in NSG mice bearing CD19-A375 melanoma tumors treated with AAVS1 control or POL2A_5()93-edited CD19 CAR T cells, measured 9 days after tumor injection. B) Percentage of CAR T cells within tumors from the mice shown in (A). C) Percentage of human CAR T cells among total CD45+cells in peripheral blood from CD 19-A375 tumor-bearing mice treated with AAVS1 control or Pol2a_5093 -edited CD 19 CAR T cells, assessed 10 days after tumor clearance.

[0029] FIGS. 12A-12B. TAF9_2303 base-edited CD19 CAR T cells enhance melanoma tumor control in vivo and exhibit increased CAR T cell infiltration in tumors. A) Tumor growth in NSG mice bearing CD19-A375 melanoma tumors treated with AAVS1 or B2M control, the positive control Regnasel edited or TAF9_2303 edited CD 19 CAR T cells. B) Percentage of CAR T cells within tumors from the mice shown in (A).

[0030] FIG. 13. POL2A_5698 and TAF8_2254 base-edited CD 19 CAR T cells potentially enhance melanoma tumor control in vivo. Tumor growth in NSG mice bearing CD19-A375 melanoma tumors treated with A AVS1 control, POL2A_5698 or TAF8_2254 base edited CD19 CAR T cells.

[0031] FIGS. 14-16. Provide the sgRNAs, the genes (accession numbers, protein sequence and chromosomal location), the edits and the effect the edit had on the T cell. In FIG. 14, for example, for amino acid numbering, the starting amino acid (such as methionine) is numbered 0 (in conventional numbering the starting amino acid begins with 1, but in, for example FIG.

[0032] 14, the starting / first amino acid is numbered 0).

[0033] DESCRIPTION

[0034] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumeratedG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0035] claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0036] The field of T cell-based therapies has demonstrated significant promise in treating oncology and autoimmune diseases by leveraging the immune system to target pathological cells. However, conventional approaches to T cell engineering, such as complete gene disruption or ectopic gene expression, often result in broad, non-specific effects that can compromise cellular viability, induce unintended immunogenicity, or fail to isolate subfunctional domains within multifunctional proteins. Additionally, traditional gene editing methods reliant on double-strand breaks (DSBs) are associated with p53-mediated responses, chromosomal rearrangements, and variable editing efficiencies across genomic loci, further limiting their therapeutic applicability. These limitations underscore the need for more precise and targeted methods to enhance T cell functionality without compromising cellular integrity or inducing off-target effects.

[0037] The present method addresses these challenges by introducing a novel approach for enhancing T cell function through base editing of genes within the RNA Pol II pre-initiation complex (PIC) and Mediator complex. Unlike conventional CRISPR-based knockout strategies, which often result in complete loss of gene function, the disclosed method utilizes base editing to introduce specific nucleotide changes that fine-tune protein functionality at the amino acid level. This enables targeted modulation of sub-functional domains within proteins, allowing for precise enhancement or suppression of specific cellular pathways. By employing high-throughput pooled screens, the method identifies amino acid embodiments that improve T cell fitness, including proliferation, IL2RA expression, cytokine production, and tumor infiltration. Furthermore, the method mitigates risks associated with DSBs, such as chromosomal instability, by utilizing base editors that operate without inducing frameshift mutations or large-scale genomic disruptions.

[0038] The disclosed concept utilizes advanced CRISPR base editing technologies, including adenine base editors (ABE) and cytosine base editors (CBE), to systematically analyze coding regions of PIC and Mediator complex genes. Through the use of sgRNA libraries and lenti viral delivery systems, the disclosed approach enables precise mutagenesis of target genes, facilitating the identification of functional hotspots that enhance T cell activity. This methodology not only improves the therapeutic potential of T cells in cancer and autoimmune disease treatment but also provides a platform for discovering novel therapeutic targets and small-molecule drugs that modulate immune cell function. By addressing the limitations ofG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0039] traditional gene editing methods, the disclosed concept represents a notable advancement in the field of immunotherapy and precision medicine.

[0040] Provide herein are methods of enhancing T cell fitness (proliferation, IL2RA expression, cytokine production) via base editor mutagenesis of the RNA Pol II pre-initiation complex (PIC) and Mediator in T cells to enhance T cell function (proliferation, IL2RA expression, cytokine production) for therapeutic use. Using CRISPR base editing, pooled screens were performed with mutagenesis of the proteins in the PIC in T cells in vitro, leading to the prioritization of amino acid variants that increase cellular abundance or IL2RA expression (IL-2receptor high affinity subunit that promotes T cell proliferation). Hits from these assays have been validated in an arrayed format to confirm changes in cell abundance and IL2RA expression. These screens were also repeated in vivo, leading to the identification of guides that result in increased tumor infiltration using a cancer model. Cells that infiltrate the tumor were also screened for IFN-y expression, leading to the identification of hits that increase or decrease IFN-y production. These screens and assays were also conducted with ch genes / proteins in Mediator in T cells.

[0041] This invention can be used in a cell therapy setting to improve T cell fitness in the treatment of cancer or autoimmune disease. Hits from the in vivo screen in particular can be used to increase cell therapy infiltration into tumors and increase IFN-y production. Small molecules that disrupt interactions highlighted by the findings can also be used as a therapy for a broad spectrum of disease.

[0042] In one embodiment, T cells are contacted with a single sgRNA targeting a specific coding region within a gene of the RNA Pol II pre-initiation complex, together with an adenine base editor delivered via a lentiviral vector and subsequently selected for enhanced IL2RA expression. In another embodiment, a population of T cells is exposed to a library of sgRNAs targeting multiple coding regions across several genes of the Mediator complex, with cytosine base editor and sgRNA delivered on separate viral vectors, and the cells are sorted based on increased cytokine production. In yet another embodiment, CD8+ T cells are modified using a combination of adenine and cytosine base editors, each encoded by distinct nucleic acid molecules, and a panel of sgRNAs targeting both PIC and Mediator complex genes, with the resulting cells selected for improved proliferation and tumor infiltration. In a further embodiment, CAR-T cells are contacted with sgRNAs targeting conserved functional domains within PIC genes, using a retroviral vector encoding the base editor, and sorted for enhanced effector function. In an additional embodiment, regulatory T cells are modified by base editing using sgRNAs with at least 85% sequence identity to those listed in Figure 15, delivered viaadeno-associated vims, and selected for altered cytokine production profiles. In another embodiment, the method is applied to allogeneic T cells, with sgRNAs and base editor delivered by plasmid DNA vectors, and the cells are sorted for increased persistence following repeated stimulation. In a further embodiment, the sgRNA and base editor are delivered together on a single viral vector to naive T cells, and the cells arc selected for enhanced proliferation and IL2RA expression. In yet another embodiment, the method is used to modify NK cells or B cells by targeting PIC or Mediator complex genes with base editors and sgRNAs, and the cells are selected for improved immune activity.

[0043] A population of cells may include T cells that have been contacted with a single sgRNA targeting a specific coding region within a gene of the RNA Pol II pre-initiation complex and an adenine base editor delivered via a lentiviral vector, with the population enriched for cells exhibiting enhanced IL2RA expression. In another embodiment, the population comprises T cells exposed to a library of sgRNAs targeting multiple coding regions across several genes of the Mediator complex, with cytosine base editor and sgRNA delivered on separate viral vectors, and the population sorted for increased cytokine production. In yet another embodiment, the population consists of CD8+ T cells modified using a combination of adenine and cytosine base editors, each encoded by distinct nucleic acid molecules, and a panel of sgRNAs targeting both PIC and Mediator complex genes, with the population selected for improved proliferation and tumor infiltration. In a further embodiment, the population includes CAR-T cells contacted with sgRNAs targeting conserved functional domains within PIC genes, using a retroviral vector encoding the base editor, and sorted for enhanced effector function. In an additional embodiment, the population comprises regulatory T cells modified by base editing using sgRNAs with at least 85% sequence identity to those listed in Figures 10 and 15, delivered via adeno-associated virus, and selected for altered cytokine production profiles. In another embodiment, the population includes allogeneic T cells with sgRNAs and base editor delivered by plasmid DNA vectors, and the cells are sorted for increased persistence following repeated stimulation. In a further embodiment, the population consists of naive T cells with sgRNA and base editor delivered together on a single viral vector, and the cells are selected for enhanced proliferation and IL2RA expression. In yet another embodiment, the population includes NK cells or B cells modified by targeting PIC or Mediator complex genes with base editors and sgRNAs, and the cells are selected for improved immune activity.

[0044] An isolated human cell may be obtained by contacting a T cell with a single sgRNA targeting a specific coding region within a gene of the RNA Pol II pre-initiation complex and an adenine base editor delivered via a lentiviral vector, with the isolated cell exhibitingenhanced IL2RA expression. In another embodiment, the isolated cell is a T cell exposed to a library of sgRNAs targeting multiple coding regions across several genes of the Mediator complex, with cytosine base editor and sgRNA delivered on separate viral vectors, and the cell is selected for increased cytokine production. In yet another embodiment, the isolated cell is a CD8+ T cell modified using a combination of adenine and cytosine base editors, each encoded by distinct nucleic acid molecules, and a panel of sgRNAs targeting both PIC and Mediator complex genes, with the cell selected for improved proliferation and tumor infiltration. In a further embodiment, the isolated cell is a CAR-T cell contacted with sgRNAs targeting conserved functional domains within PIC genes, using a retroviral vector encoding the base editor, and sorted for enhanced effector function. In an additional embodiment, the isolated cell is a regulatory T cell modified by base editing using sgRNAs with at least 85% sequence identity to those listed in Figures 10 and 15, delivered via adeno-associated virus, and selected for altered cytokine production profiles. In another embodiment, the isolated cell is an allogeneic T cell with sgRNAs and base editor delivered by plasmid DNA vectors, and the cell is selected for increased persistence following repeated stimulation. In a further embodiment, the isolated cell is a naive T cell with sgRNA and base editor delivered together on a single viral vector, and the cell is selected for enhanced proliferation and IL2RA expression. In yet another embodiment, the isolated cell is an NK cell or B cell modified by targeting PIC or Mediator complex genes with base editors and sgRNAs, and the cell is selected for improved immune activity.

[0045] An isolated cell may be generated by base editing a gene listed in Figures 10 and 14-16), resulting in altered cellular activities. In one embodiment, the isolated cell is a T cell with a base edited gene in the RNA Pol II pre-initiation complex, exhibiting increased IL2RA expression. In another embodiment, the isolated cell is a CD8+ T cell with a base edited gene in the Mediator complex, showing improved proliferation and tumor infiltration. In yet another embodiment, the isolated cell is a CAR-T cell with a base edited gene in a conserved functional domain of the PIC, demonstrating enhanced effector function. In a further embodiment, the isolated cell is a regulatory T cell with a base edited gene in the Mediator complex, displaying altered cytokine production profiles. In an additional embodiment, the isolated cell is an allogeneic T cell with a base edited gene in Figures 10 and 14-16, selected for increased persistence following repeated stimulation. In another embodiment, the isolated cell is a naive T cell with a base edited gene in Figures 10 and 14-16, selected for enhanced proliferation and IL2RA expression. In yet another embodiment, the isolated cell is an NK cell or B cell with a base edited gene in Figures 10 and 14-16, selected for improved immune activity.In one embodiment, a composition comprising a plurality of isolated cells, such as T cells with base edited genes in the RNA Pol II pre-initiation complex, is administered to a mammal to prevent, inhibit, or treat cancer, with the cells exhibiting increased IL2RA expression and enhanced proliferation. In another embodiment, the composition includes CD8+ T cells with base edited genes in the Mediator complex, selected for improved tumor infiltration and cytokine production, and is systemically administered to a human patient. In yet another embodiment, the composition comprises CAR-T cells with base edited genes in conserved functional domains of the PIC, demonstrating enhanced effector function, and is used to treat sarcoma, lymphoma, melanoma, or carcinoma. In a further embodiment, the composition contains allogeneic T cells with base edited genes delivered by plasmid DNA vectors, selected for increased persistence following repeated stimulation, and administered intravenously to treat immune cell cancers. In an additional embodiment, the composition includes regulatory T cells or naive T cells with base edited genes in Figures 10 and 14-16, selected for altered cytokine production profiles or enhanced proliferation, and is used for the treatment of lung cancer, breast cancer, prostate cancer, pancreatic cancer, or ovarian cancer. In yet another embodiment, the composition comprises NK cells or B cells with base edited genes in Figures 10 and 14-16, selected for improved immune activity, and is administered to augment anti-tumor responses in a mammalian subject.

[0046] In one embodiment, a composition comprising a plurality of isolated immune cells, such as T cells with base edited genes in the RNA Pol II pre-initiation complex, is administered to a mammal to prevent, inhibit, or treat an autoimmune disease, with the cells exhibiting increased IL2RA expression and enhanced proliferation. In another embodiment, the composition includes regulatory T cells with base edited genes in the Mediator complex, selected for altered cytokine production profiles, and is systemically administered to a human patient. In yet another embodiment, the composition comprises CD4+ or CD8+ T cells with base edited genes in conserved functional domains of the PIC or Mediator complex, demonstrating enhanced effector function or altered immune activity, and is used to treat autoimmune conditions such as multiple sclerosis. In a further embodiment, the composition contains allogeneic T cells with base edited genes delivered by plasmid DNA vectors, selected for increased persistence following repeated stimulation, and administered intravenously to treat autoimmune diseases. In an additional embodiment, the composition includes naive T cells with base edited genes in Figures 10 and 14-16, selected for enhanced proliferation or altered cytokine production, and is used for the treatment of autoimmune disorders. In yet another embodiment, the composition comprises NK cells or B cells with base edited genes inFigures 10 and 14-16, selected for improved immune regulation, and is administered to modulate immune responses in a mammalian subject.

[0047] Definitions

[0048] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 16th Edition, by M. Larranga, R Lewis Sr., and R Lewis, New York, N.Y., 2016.

[0049] References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0050] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0051] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is di- substituted.

[0052] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a listing of items, “and / or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0053] As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising."

[0054] The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the endpoints of a recited range as discuss above in this paragraph.

[0055] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

[0056] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals,G L2025-001 / / SF2O25-143 / / 3730.239WO1

[0057] substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

[0058] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group.

[0059] Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0060] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0061] The use of the word “detect” and its grammatical variants refers to measurement of the species without quantification, whereas use of the word “determine” or “measure” with their grammatical variants are meant to refer to measurement of the species with quantification. The terms “detect” and “identify” are used interchangeably herein.

[0062] The term “standard,” as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function. Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.

[0063] The terms “comprises,” “comprising,” and the like can have the meaning ascribed to them in U.S. Patent Law and can mean “includes,” “including” and the like. As used herein, “including” or “includes” or the like means including, without limitation.The term “guide RNA” as used herein refers to either a single guide RNA (sgRNA) or a crRNA (spacer). In some cases, the at least one sgRNA has a sequence with at least 95% sequence identity to any of guide RNAs shown in the Figures / Tables filed herewith. In some cases, at least sgRNA has a sequence such as any of the guide RNAs in the Figures, or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, or a combination thereof. In some cases, cells can be incubated with one or two or more sgRNAs described herein.

[0064] A "vector" or “delivery” vehicle refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide or polypeptide, and which can be used to mediate delivery of the polynucleotide or polypeptide to a cell or intercellular space, either in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, nanoparticles, or microparticles and other delivery vehicles. In one embodiment, a polynucleotide to be delivered, sometimes referred to as a "target polynucleotide" or "transgcnc," may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest), a coding sequence of interest and / or a selectable or detectable marker.

[0065] "Transduction," "transfection," "transformation" or "transducing" as used herein, are terms referring to a process for the introduction of an exogenous polynucleotide into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell, and includes the use of recombinant virus to introduce the exogenous polynucleotide to the host cell. Transduction, transfection or transformation of a polynucleotide in a cell may be determined by methods well known to the art including, but not limited to, protein expression (including steady state levels), e.g., by ELISA, flow cytometry and Western blot, measurement of DNA and RNA by hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays. Methods used for the introduction of the exogenous polynucleotide include well-known techniques such as viral infection or transfection, lipofection, transformation and electroporation, as well as other non-viral gene delivery techniques. The introduced polynucleotide may be stably or transiently maintained in the host cell.

[0066] "Gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.

[0067] "Gene transfer" refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene.G L2025-001 / / SF2O25-143 / / 3730.239WO1

[0068] "Gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification.

[0069] An "infectious" virus or viral particle is one that comprises a polynucleotide component which is capable of delivering into a cell for which the viral species is trophic. The term does not necessarily imply any replication capacity of the virus.

[0070] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single- stranded forms known or predicted to make up the double-stranded form.

[0071] A "transcriptional regulatory sequence" refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences of use generally include at least one transcriptional promoter and may also include one or more enhancers and / or terminators of transcription.

[0072] "Operably linked" refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner. By way of illustration, a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence. An operably linked TRS is generally joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.

[0073] "Heterologous" means derived from a genotypically distinct entity from the entity to which it is compared. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, a transcriptional regulatory element such as a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcriptional regulatory element.

[0074] A "terminator" refers to a polynucleotide sequence that tends to diminish or prevent read-through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator). The degree to which transcription is disrupted is typically a function of the base sequence and / or the lengthG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0075] of the terminator sequence. In particular, as is well known in numerous molecular biological systems, particular DNA sequences, generally referred to as "transcriptional termination sequences" are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and / or disengage from the DNA being transcribed. Typical examples of such sequence- specific terminators include polyadenylation ("polyA") sequences, e.g., SV40 poly A. In addition to or in place of such sequence-specific terminators, insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated. Terminators may thus prevent transcription from only one direction ("uni-directional" terminators) or from both directions ("bi-directional" terminators) and may be comprised of sequence-specific termination sequences or sequence-non-specific terminators or both. A variety of such terminator sequences are known in the art; and illustrative uses of such sequences within the context of the present disclosure are provided below.

[0076] "Host cells," "cell lines," "cell cultures," "packaging cell line" and other such terms denote higher eukaryotic cells, such as mammalian cells including human cells, useful in the present disclosure, e.g., to produce recombinant vims or recombinant polypeptide. These cells include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.

[0077] "Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant vims is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide constmct and progeny of the original vims constmct.

[0078] A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process,G L2025-001 / / SF2O25-143 / / 3730.239WO1

[0079] and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. Promoters include AAV promoters, c.g., P5, Pl 9, P40 and AAV ITR promoters, as well as heterologous promoters.

[0080] An "expression vector" is a vector comprising a region which encodes a gene product of interest and is used for effecting the expression of the gene product in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0081] The terms "polypeptide" and "protein" arc used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component.

[0082] An "isolated" polynucleotide, e.g., plasmid, vims, polypeptide or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Isolated nucleic acid, peptide or polypeptide is present in a form or setting that is different from that in which it is found in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. The isolated nucleic acid molecule may be present in single-stranded or doublestranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the molecule will contain at a minimum the sense or coding strand (i.e., the molecule may single- stranded) but may contain both the sense and anti-sense strands (i.e., the molecule may be double-stranded). Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. For example, a 2-fold enrichment, 10-fold enrichment, 100-fold enrichment, or a 1000-fold enrichment.G L2025-001 / / SF2O25-143 / / 3730.239WO1

[0083] A “transcriptional regulatory sequence’’ refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences of use generally include at least one transcriptional promoter and may also include one or more enhancers and / or terminators of transcription.

[0084] “Conservative’’ amino acid substitutions arc, for example, aspartic-glutamic as polar acidic amino acids; lysine / arginine / histidine as polar basic amino acids; leucine / isoleucine / methionine / valine / alanine / glycine / proline as non-polar or hydrophobic amino acids; serine / threonine as polar or uncharged hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the polypeptide. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic; trp, tyr, phe.

[0085] The disclosure also envisions polypeptides with non-conservative substitutions. Nonconservative substitutions entail exchanging a member of one of the classes described above for another.

[0086] As used herein, "individual" (as in the subject of the treatment) means a mammal. Mammals include, for example, humans; non-human primates, e.g.. apes and monkeys; and non-primates, e.g., dogs, cats, rats, mice, cattle, horses, sheep, and goats. Non-mammals include, for example, fish and birds.

[0087] "Substantially" as the term is used herein means completely or almost completely; for example, a composition that is "substantially free" of a component either has none of the component or contains such a trace amount that any relevant functional property of thecomposition is unaffected by the presence of the trace amount, or a compound is "substantially pure" is there are only negligible traces of impurities present.

[0088] "Treating" or "treatment" within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, "inhibiting" means inhibition of further progression or worsening of the symptoms associated with the disorder or disease, and "preventing" refers to prevention of the symptoms associated with the disorder or disease.

[0089] As used herein, an "effective amount" or a "therapeutically effective amount" of an agent, refers to an amount of the agent that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms (e.g., ameliorate one or more symptoms), or prevents or provides prophylaxis for the disorder or condition, e.g., an amount that is effective to prevent, inhibit or treat in the individual one or more symptoms.

[0090] In particular, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent(s)are outweighed by the therapeutically beneficial effects.

[0091] The term "sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or there above), e.g., between about 100 and 1,000 nucleotides in length (or any integer therebetween), e.g., between about 200 and 500 nucleotides in length.

[0092] For example, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (e.g., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.The term "exogenous," when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell. By way of a non-limiting example, an exogenous nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different nucleic acid sequence than that found in nature, e.g., an expression cassette which links a promoter from one gene to an open reading frame for a gene product from a different gene.

[0093] "Transformed" or "transgenic" is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or infection with a recombinant viral vector.

[0094] The term "sequence homology" means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, or 6 bases or less or 2 bases or less. When using oligonucleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); not less than 9 matches out of 10 possible base pair matches (90%), or not less than 19 matches out of 20 possible base pair matches (95%).

[0095] Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less or 2 or less. Alternatively, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. The two sequences or parts thereof are more homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.The term "corresponds to" is used herein to mean that a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion of a reference polypeptide sequence, e.g., they have at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97% or more, e.g., 99% or 100%, sequence identity. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".

[0096] The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, e.g., at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

[0097] As used herein, "substantially pure" or "purified" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), for instance, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, or more than about 85%, about 90%, about 95%, and about 99%. The object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0098] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Molecular Cloning: A Laboratory Manual, 4th cd., Green and Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2014; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981.

[0099] RNA Pol II pre-initiation complex (PIC) and the Mediator complex

[0100] RNA Pol II pre-initiation complex (PIC) and the Mediator complex are components of the transcription machinery in eukaryotic cells, playing roles in the initiation of transcription by RNA polymerase II (Pol II).

[0101] RNA Pol II Pre-initiation Complex (PIC)

[0102] PIC is a large assembly / complex of proteins (about 80) that assembles at the promoter region of a gene to initiate transcription. It positions RNA polymerase II at the transcription start site and facilitates the unwinding of DNA to allow transcription to begin. PIC includes RNA polymerase II and several general transcription factors (GTFs), such as TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH. These factors recruit RNA Pol II to the promoter and aid in the formation of a transcriptionally competent complex. Assembly of the PIC is a stepwise process, beginning with the binding of TFIID to the TATA box in the promoter region, followed by the sequential addition of other GTFs and RNA Pol II.

[0103] Mediator Complex

[0104] The Mediator complex is a multi-protein complex (of about 30 proteins) that acts as a bridge between PIC and regulatory proteins, such as transcriptional activators and repressors. It plays a role in regulating gene expression by transmitting signals from these regulatory proteins to the transcription machinery. The Mediator complex is composed of multiple subunits, which can vary depending on the organism and the specific gene being transcribed. It is organized into several modules, including the head, middle, tail, and kinase modules. The Mediator complex facilitates the recruitment of RNA Pol II to the promoter, stabilizes PIC, and helps in the transition from transcription initiation to elongation. It also integrates signals from various signaling pathways to modulate transcription in response to cellular and environmental cues.G L2025-001 / / SF2O25-143 / / 3730.239WO1

[0105] Provided herein is the genetic manipulation, through base editing, of the RNA Pol II pre-initiation complex and Mediator to enhance T cell function and improve their fitness and effectiveness in therapeutic applications such as cancer treatment.

[0106] Base Editing

[0107] Base Editing (BE) is a method that, guided by CRISPR-Cas9, introduces specific A to G or C to T mutations in the genomic DNA without double-strand breaks. This method has traditionally been used to correct specific pathologic mutations with increased on-target fidelity and minimized translocation risk. More recently, base editing was used to introduce loss of-function mutations in primary human T cells to create functional knockouts. While Cytidine base editors (CBE) can directly introduce stop codons by converting amino acid coding triplets into TGA, TAA or TAG, ABE and CBE both can cause functional knockouts by altering splice sites (to induce frame shift) or removing start codons. These approaches have successfully been used to introduce multiplexed knockouts in primary human T cells. It was hypothesized that base editing could be used for functional interrogation of proteins at amino acid level resolution.

[0108] For example, one or more target regions in one or more selected genes are identified (e.g., identified by an editing window spanning a portion of the sgRNA length including bases directly upstream of the 5’ end of the sgRNA) that can be modified to alter gene function (e.g., to fine tune in a positive or negative manner). In one embodiment, the PIC and Mediator genes play roles in T cell biology or are members of families of genes involved in T cell / lymphocyte biology. In one embodiment, all available sgRNAs with an NGG PAM sequence in the editing window being (at least in part) in the coding regions of these genes were prepared, thus tiling the entire coding region of the PIC and Mediator complex genes. Since this was not a traditional CRISPR knockout screen, but a base editing screen, the chosen approach allowed one to mutate specific parts of these genes without causing frameshift mutations. Further, given the broad variety of sgRNAs, the information provided by the screen allows for the fine tuning of the function of respective gene product (in comparison to traditional knockout where it is an all-or-nothing mutation). Some of the hotspot regions that were identified may be used to introduce the opposite effect a traditional knockout would have (e.g., enhance protein function). Furthermore, this approach allows targeting of specific functions within the overall functionality the respective protein. Thus, the identified sgRNAs can be employed to modify any cell that expresses the target gene or to identify functional or targetable regions within the respective protein independent of its cellular expression pattern. More broadly, the screens define a targetable region in these genes other than that for the specific sgRNAs employedG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0109] because of the absence of control for introduced amino acid changes that occur at a defined pattern (within the editing window, A becomes G or C becomes T). Thus, sgRNAs were used to define gene regions that, when altered, influence gene function (decrease or enhance).

[0110] The sgRNAs may be employed to modify gene / protein function, for example, alter but not eliminate the function(s), in cells including but not limited to Pan T cells, CD4 T cells, CD8 T cells, regulatory T cells, NK cells, B cells or other lymphoid cells to also alter their function in a therapeutic setting (enhance activity or decrease specific activity). The identified protein modification of region allowing for modified protein functionality could be used in any context this gene or protein might be expressed, including other cell types or as a synthetic approach. For example, cells not naturally expressing the protein of interest could be modified by expression of an identified mutant of that protein to alter cellular functions. In another example, the modified protein could be incorporated in other synthetic therapies, e.g., anti-cancer or anti-autoimmune T cells.

[0111] Exemplary Screening Methods

[0112] Previous CRISPR-enabled screening has primarily focused on whole gene inhibition / knockout or activation, whereas a high-resolution method can identify functionally distinct regions within a gene, which can be of therapeutic relevance. When combined with large scale phenotypic screening approaches, the effects of amino acid level disruption across hundreds of genes involved in T cell activity can rapidly be determined. This high throughput examination enables quick identification of functionally distinct regions - regions that performed better or orthogonally to inhibition / activation of the entire gene - that could also be used for therapeutic applications. For example, genes and coding regions may be identified to more finely tuned T cell responses; sgRNA may be identified for base editing mediated knockout, alleviating many concerns with standard CRISPR knockout; or loss / gain of function mutations into specific functional regions of a protein may be identified, allowing dissection of different functions of a single protein.

[0113] To optimize this technology for screening in primary human T cells, the efficiency of a variety of base editors on specific genes was tested. Lentiviral transfer plasmids of the second generation were modified to enhance the titer. With these constructs, highly efficient edits were produced with base editor and sgRNA delivered on separated viruses. By adding a combination of blasticidin and puromycin to these constructs, successfully transduced cells were enriched, yielding a pure population of edited cells. Having established the delivery and functionality of the base editor machinery in primary human T cell for pooled screening (it was found that the lentiviral approach was superior to a hybrid sgRNA lentivirus approach + base editor mRNA),an sgRNA library was prepared for the coding region of PIC and Mediator complex genes involved in T cell activity. T cells were sorted for their enhanced T cell function (proliferation, IL2RA expression, cytokine production). T cells were also subjected to repeated stimulation to test which sgRNA mediated mutations provide a proliferative advantage compared to control cells. sgRNAs and groups of sgRNAs which mapped to distinct regions within target genes and caused gain-or-loss of T cell activity were identified. Notably, these effects frequently exceeded the effects of knockout / activation or caused the opposite effect of knockout / activation -highlighting the power of base editing to introduce functional changes to genes which are otherwise not possible. sgRNAs were found that mediate negative effects as well as sgRNAs mediating positive effects within the same gene, using a single editing method to cause opposite effects. Consequently, the findings were validated by assessing T cell activation in arrayed format with single sgRNAs targeting representative regions within a selected set of genes. It was also shown that some of the introduced perturbations increased the cancer killing potency of T cells.

[0114] In summary, a highly efficient method to perform pooled base editing screens in primary human T cells was identified. This includes but is not limited to ABE, CBE and with different types of Cas9 (Cas9 with NGG PAM, SpG Cas9 with NG PAM, or NG Cas9 with NG PAM). This approach was used to identify genomic regions (at the amino acid level) in coding regions which can be targeted to alter T cell functionality in multiple contexts. Thus, methods and compositions to generate base edited T cells with improved functionality that could enhance immunotherapies are provided. The functionality includes direct responses to T cell activation as well as the integration of environmental factors. A set of screens identified causal relationships between mutation sites in the entire coding regions of selected genes and their effect on T cell activation and function. Thus, the methods also provide a discovery platform to identify therapeutic targets.

[0115] In one embodiment, a CBE can be employed. In one embodiment, the CBE is a first-generation base-editor (CBE1), one fusing a rat-derived cytosine deaminase Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 1 (APOBEC1) to the amino terminus of catalytically deficient, or “dead”, Cas9 (dCas9). In a narrow window of the non-targeted strand, CBE1 deaminates cytosine to uracil. Uracil is then recognized by cell replication machinery as a thymine, resulting in a C-G to T-A transition. In one embodiment, the base editor is a second-generation cytosine base-editor (CBE2), e.g., one prepared by fusing an uracil DNA glycosylase inhibitor (UGI) to the C-terminus of BE1, inhibiting the activity of UDG. BE3 was developed by restoring histidine at position 840 (H840, HNH catalytic domain) in dCas9 toG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0116] generate a base-editor that uses Cas9 nickase (nCas9). This variant induces a nick in the G-containing strand of the U-G intermediate (non-edited DNA strand) to bias cellular repair of the intermediate towards a U-A outcome, further converted to T- A during DNA replication. In one embodiment, the base editor is a fourth-generation cytosine base-editor (CBE4), e.g., generated by fusing an additional copy of UGI to the N terminus of nCas9 with an optimized 27 bp linker. YEE-BE3 was developed by screening several mutations previously reported to modulate the catalytic activity of cytosine deaminases in the APOBEC family to generate an improved r APOBEC 1 with a narrower editing window and reduced “bystander editing” compared to CBE3. Gam, a DNA-binding protein from bacteriophage Mu, can form a complex with free-ends of DBSs, thus preventing NHEJ-mediated repair and reducing indel formation. These changes resulted in BE4-Gam, which is characterized by higher base-editing efficiency, increased product purity, and decreased indel frequency. Two nuclear localization signals (NLS) were added to nCas9 and after codon-optimization and ancestral sequence reconstruction on APOBEC, yielding BE4max and ancBE4max. Another base-editing system, Target-AID (activation-induced cytidine deaminase), was developed and composed of nCas9, Petromyzon marinus cytidine deaminase 1 (pmCDAl), which is similar to rAPOBECl in structure and function. The use of alternative cytosine deaminase enzymes yields base editors with alternative sequence motif preference and the ability to efficiently edit methylated cytosines. Most recently, Liu and colleagues used phase assisted continuous evolution (PACE) to evolve CBEs and generate evoAPOBECl-BE4max, which can efficiently edit cytosine in G / C sequences (a disfavored context for wild-type APOBEC1 deaminase) and evoFERNY-BE4max, a smaller deaminase that edits efficiently in all tested sequence contexts.

[0117] Base-editors incorporating different CRISPR-associated nuclease enzymes are also envisioned for use in the method. CBEs based on SpCas9 are limited by their G / C-rich PAM sequence. In order to expand the scope of base-editing, Li et al. generated a Cpfl -based cytosine deaminase base-editor by fusing catalytically inactive LbCpf 1 (dLbCpf 1 ) or dAsCpf 1 with rAPOBECl and UGI (creating dLbCpf 1-BEO and dAsCpfl-BEO). A variety of engineered Cas9 variants with altered PAM sequences and improved cleavage specificity have been developed and may allow for further expansion of the targeting scope of CRISPR-base-editing reagents.

[0118] Adenine base-editors induce A to G conversions. The ABE-dCas9 fusion binds to a target DNA sequence in a guide RNA-programmed manner, and the deoxyadenosine deaminase domain catalyzes an adenine to inosine transition. In the context of DNA replication, inosine is interpreted as guanine, and the original A-T base pair may be replaced with a G-Cbase pair at the target site. Escherichia coli tRNA adenosine deaminase, TadA (ecTadA) converts adenine to inosine in the single- stranded anticodon loop of tRNAARG, and shares sequence similarity with the APOBEC family. The first-generation adenine base-editors (ABE1.2) was generated by fusing the evolved TadA variant (TadA*) to the N-terminus of nCas9 through XTEN (a 16 amino acid linked used in BE3), with the C terminal of nCas9 fused with a nuclear localization signal (TadA*-XTEN-nCas9-NLS). In comparison with cytosine base-editing, adenine base-editing by ABE yields a much cleaner product that has virtually no indels, and there are no reports of significant off-target (A-to-non-G) edits to date.

[0119] To improve ABEs, a single-chain heterodimer was prepared comprised of a wild-type non-catalytic TadA monomer and evolved ecTadA monomer (TadA-TadA*). To improve editing efficiency, further optimization of ABE was performed. Extensive PACE and protein engineering resulted in seventh generation ABEs (ABE7.10), which converted target A-T to G-C efficiently (-50%) in human cells. Only about one-quarter of pathogenic transition mutations encompass an appropriately located NGG PAM site that facilitates SpCas9-mcdiatcd baseediting. Unlike CBEs, which have proven to be broadly customizable with many Gas orthologs, ABEs have shown limited compatibility with Cas9 of any origin other than SpCas9. Some homologs such as SaCas9 and circularly permuted Cas9 (CP-Cas9) have been adapted. Phage-assisted continuous and non-continuous evolution (PACE and PANCE) methods were used to enhance the catalytic rate of the deoxy adenosine deaminase enzyme by 590-fold compared to that of ABE7.10. The next generation of ABEs, designated ABE8e, shows greatly enhanced activity and compatibility with diverse Cas9 homologs. As expected, the targeting scope of ABE8e also increased off-target RNA and DNA editing.

[0120] Exemplary base editors useful in the screening methods include but are not limited to ABE8e, BE1, BE2, HF2-BE2, BE3, HF-BE3, YE1-BE3, EE-B3, YEE-BE3, VQR-BE3, EQR-BE3, VRER-BE3, SaKKHBE3, FNLS-BE3, RA-BE3, A3A-BE3, eA3A-HFl-BE3-2xUGI, eA3A-Hypa-BE3-2xUGI, hA3A-BE3, hA3B-BE3, hA3G-BE3, hAID-BE3, SaCas9-BE3, xCas9-BE3, ScCas9-BE3, SniperCas9-BE3, iSpyMac-BE3, Target-AID, Target-AID-NG, CRISPR-X, TAM, BE-PLUS, BE4, BE4-Gam, BE4-Max, AncBE4-Max, SaCas9-BE4, SaCas9-BE4-Gam, evoBE4max, evoFERNY-BE4max, Casl2a-BE, ABE7.8 / 9 / 10, xCas9-ABE7.10, VQR-ABE, Sa(KKH)-ABE, ABEmax, ABE7.10max, ABE8e, PEI, PE2, or PE3.

[0121] In other screens, small molecule drugs targeting one of the identified functional regions can be identified that allow for enhanced or decreased immune activity (in a variety of immune related pathologies including cancer, autoimmunity, and the like).G L2025-001 / / SF2O25-143 / / 3730.239WO1

[0122] The use of the method can provide for identification and targeting of protein subfunctions (e.g., higher resolution than overall loss- or gain- of function methods), tools to identify targetable structures of proteins, and identification of hits and functional regulators for exploitation as therapeutic targets.

[0123] The use of a method for high throughput base editing screens in primary human T cells using viruses, such as lentivirus, provides for an approach for high throughput screening of protein / gene domains at amino acid level, in primary human T cells and may result in a large set of previously uncharacterized targets that alter T cell function, base edited T cells with enhanced functionality and the ability to dissect different (including opposing) functional regions within a protein and exploitation for therapeutic or diagnostic purposes.

[0124] The methods and resulting composition provide for rapid discovery, including discovery of variants or mutations that affect cell functions in patients, and therapeutic applicability of targets for immune cell therapies, highly multiplexable and seamless integration with other gain- or loss-of function strategics, loss and gain of protein (sub-) functions, e.g., using a single editing method, multiplexable and / or within a single protein, identification of sgRNAs to mediate functional base editor knockouts of desired genes, and discovery and targeting of protein (e.g. domain or amino acid specific) subfunctions.

[0125] Exemplary Cells for Modification

[0126] In one embodiment, the cells that are modified with sgRNAs or the genome of which is modified to have the modified sites described herein or expresses a protein having one or more of the modifications disclosed herein, are immune cells. In one embodiment, the cells are T cells. In one embodiment, the cells are CD4+ cells. In one embodiment, the cells are CD8+ cells. In one embodiment, the cells are CAR-T cells. In one embodiment, the icells are naive T cells, stem cell memory cells, T SCM: T Central Memory cells, T CM; T effector memory cells, T EM; or T effector cells, T EFF. In one embodiment, the cells are Th (T helper) 1 , Th2, Th9, Thl7, Th22, Treg (regulatory T cells), or Tfh (follicular helper T cells). In one embodiment, the cells are regulatory T cells, NK cells, or B cells.

[0127] Exemplary Therapeutic Uses of Modified Cells

[0128] Ex vivo or in vivo genome edited immune cells can be employed for therapeutic purposes. In addition, synthetic constructs that alter immune cell function (e.g., by incorporating or using functional proteins or protein domains that are identified by the screening method) may be employed for therapeutic purposes.

[0129] Immune cells modified as described herein may be employed in a method to prevent, inhibit or treat an autoimmune disease. In one embodiment, cells of a mammal may be obtainedG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0130] and modified as described herein and reintroduced to the mammal to, for example, provide activated / enhanced T cell activity, suppress an immune function in the mammal, thereby alleviating one or more symptoms of the autoimmune disease. Autoimmune diseases within the scope of this disclosure include but are not limited to rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto’s thyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune-associated infertility, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), bullous pemphigoid, Sjogren's syndrome, insulin resistance, and autoimmune diabetes mellitus (type 1 diabetes mellitus; insulin-dependent diabetes mellitus). In one embodiment, the autoimmune disease is multiple sclerosis (MS), systemic sclerosis (SSc), type 1 diabetes (T1D), Grave's disease (GD), systemic lupus erythematosus (SLE), aplastic anemia (AA), or vitiligo.

[0131] Immune cells modified as described herein may be employed in a method to prevent, inhibit or treat cancer. In one embodiment, cells of a mammal may be obtained and modified as described herein and reintroduced to the mammal to, for example, augment an immune function in the mammal, thereby alleviating one or more symptoms of the cancer. Cancers within the scope of this disclosure include but are not limited to carcinomas (e.g., squamouscell carcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cell carcinomas), particularly those of the bladder, bone, bowel, breast, cervix, colon (colorectal), esophagus, head, kidney, liver (hepatocellular), lung, nasopharyngeal, neck, ovary, pancreas, prostate, and stomach; leukemias, such as acute myelogenous leukemia, acute lymphocytic leukemia, acute promyelocytic leukemia (APL), acute T-cell lymphoblastic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, neutrophilic leukemia and stem cell leukemia; benign and malignant lymphomas, particularly Burkitt's lymphoma, Non-Hodgkin's lymphoma and B-cell lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi’s sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; tumors of the central nervous system (e.g., gliomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas, neuroblastomas,G L2025-001 / / SF2O25-143 / / 3730.239WO1

[0132] ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas); germ-line tumors (e.g., bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer (e.g., small cell lung cancer, mixed small cell and non-small cell cancer, pleural mesothelioma, including metastatic pleural mesothelioma small cell lung cancer and non-small cell lung cancer), ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma; mixed types of neoplasias, particularly carcinosarcoma and Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumor and teratocarcinomas, among others.

[0133] Preparation of Expression Cassettes

[0134] To prepare expression cassettes having sequences encoding a base editor and / or sgRNAs, e.g., disclosed herein, or both, for transformation, the recombinant DNA sequence or segment may be circular or linear, double- stranded or single-stranded. A DNA sequence which encodes an RNA sequence that is substantially complementary to a mRNA sequence encoding a gene product of interest is typically a "sense" DNA sequence cloned into a cassette in the opposite orientation (i.e., 3' to 5' rather than 5' to 3’). Generally, the DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the DNA in a cell. As used herein, "chimeric" means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the "native" or wild type of the species.

[0135] Aside from DNA sequences that serve as transcription units, or portions thereof, a portion of the DNA may be untranscribed, serving a regulatory or a structural function. For example, the DNA may itself comprise a promoter that is active in eukaryotic cells, e.g., mammalian cells, or in certain cell types, or may utilize a promoter already present in the genome that is the transformation target of the lymphotropic virus. Such promoters include the CMV promoter, as well as the SV40 late promoter and retroviral LTRs (long terminal repeat elements), although many other promoter elements well known to the art may be employed, e.g., the MMTV, RSV, MLV or HIV LTR. In one embodiment, expression is inducible.

[0136] In one embodiment, a tissue-specific promoter (or enhancer) is employed.

[0137] Other elements functional in the host cells, such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the recombinant DNA. Such elements may or may not be necessary for the function of the DNA but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may beG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0138] included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.

[0139] The recombinant DNA to be introduced into the cells may contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfi; bar, aroA, puro, hyg, dapA and the like. See also, the genes listed on Table 1 of Lundquist et al. (U.S. Patent No. 5,848,956).

[0140] Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins arc well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Exemplary reporter genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the betaglucuronidase gene (gus) of the uidA locus of E. coli, the green, red, or blue fluorescent protein gene, and the luciferase gene. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

[0141] The general methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful herein.

[0142] The recombinant DNA can be readily introduced into the host cells, e.g., mammalian cells, such as immune cells, by transfection with the DNA or the corresponding RNA, or infection with a virus having an expression vector comprising the recombinant DNA, by any procedure useful for the introduction into a particular cell, e.g., physical or biological methods, to yield a transformed (transgenic) cell having the recombinant DNA so that the DNA sequence of interest is expressed by the host cell. In one embodiment, the recombinant DNA is stably integrated into the genome of the cell.

[0143] Physical methods to introduce a recombinant DNA into a host cell include calcium-mediated methods, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods to introduce the DNA of interest into a host cell include the use of DNA and RNA viral vectors. Viral vectors, e.g., retroviral or lentiviral vectors, have becomeG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0144] a widely used method for inserting genes into eukaryotic cells, such as mammalian, e.g., human cells. Other viral vectors can be derived from poxviruses, e.g., vaccinia viruses, herpes viruses, adenoviruses, adeno-associated viruses, baculoviruses, and the like.

[0145] To confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular gene product, e.g., by immunological means (ELIS As and Western blots) or by other molecular assays.

[0146] To detect and quantitate RNA produced from introduced recombinant DNA segments, RT-PCR may be employed. In this application of PCR, it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique demonstrates the presence of an RNA species and gives information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and only demonstrate the presence or absence of an RNA species.

[0147] While Southern blotting and PCR may be used to detect the recombinant DNA segment in question, they do not provide information as to whether the recombinant DNA segment is being expressed. Expression may be evaluated by specifically identifying the peptide products of the introduced DNA sequences or evaluating the phenotypic changes brought about by the expression of the introduced DNA segment in the host cell.

[0148] Vectors or Vehicles for Delivery

[0149] Delivery vectors or vehicles include, for example, viral vectors, microparticles, nanoparticles, liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a gene, sgRNA or a protein to a host cell, e.g., a gene to provide for recombinant expression of a polypeptide encoded by the gene. Vectors or vehicles can also comprise other components or functionalities that further modulate gene delivery and / or gene expression, or that otherwise provide beneficial properties. Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector by the cell; components that influence localization of the transferred gene within the cell after uptake (such as agents mediating nuclear localization);and components that influence expression of the gene. Such components also might include markers, such as detectable and / or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector or have taken up protein delivered by a vehicle. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated. A variety of such marker genes have been described, including bifunctional (i.e., positive / negative) markers (see, e.g., WO 92 / 08796; and WO 94 / 28143). Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts. A large variety of such vectors are known in the art and are generally available.

[0150] Vectors or vehicles within the scope of the disclosure include, but arc not limited to, isolated nucleic acid, e.g., plasmid-based vectors which may be extra-chromosomally maintained, and viral vectors, e.g., recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus, including viral and non-viral vectors, or proteins which are present in liposomes, e.g., neutral or cationic liposomes, such as DOSPA / DOPE, DOGS / DOPE or DMRIE / DOPE liposomes, and / or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA / DOPE) complexes. Vectors or vehicles may be administered via any route including, but not limited to, intramuscular, buccal, rectal, intravenous or intracoronary administration, and transfer to cells may be enhanced using electroporation and / or iontophoresis. In one embodiment, vectors are locally administered.

[0151] In one embodiment, an isolated polynucleotide or vector having that polynucleotide, encoding a polypeptide or fusion protein that has substantial identity, e.g., at least 80% or more, e.g., 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99% and up to 100%, amino acid sequence identity to a protein encoded by one of the genes disclosed herein, or a portion thereof, is envisioned.

[0152] Retroviral vectors

[0153] Retroviral vectors exhibit several distinctive features including their ability to stably and precisely integrate into the host genome providing long-term transgene expression. These vectors can be manipulated ex vivo to eliminate infectious gene particles to minimize the riskof systemic infection and patient-to-patient transmission. Pseudotyped retroviral vectors can alter host cell tropism.

[0154] Lentiviruses

[0155] Lentiviruses are derived from a family of retroviruses that include human immunodeficiency virus and feline immunodeficiency virus. However, unlike rclro viruses that only infect dividing cells, lentiviruses can infect both dividing and nondividing cells. Although lentiviruses have specific tropisms, pseudotyping the viral envelope with vesicular stomatitis virus yields virus with a broader range (Schnepp et al., Meth, MoL Med., 69:427 (2002)).

[0156] Adenoviral vectors

[0157] Adenoviral vectors may be rendered replication-incompetent by deleting the early (El A and E1B) genes responsible for viral gene expression from the genome and are stably maintained into the host cells in an extrachromosomal form. These vectors have the ability to transfect both replicating and nonreplicating cells and, in particular, these vectors have been shown to efficiently infect cardiac myocytes in vivo, c.g., after direction injection or perfusion. Adenoviral vectors have been shown to result in transient expression of therapeutic genes in vivo, peaking at 7 days and lasting approximately 4 weeks. The duration of transgene expression may be improved in systems utilizing neural specific promoters. In addition, adenoviral vectors can be produced at very high titers, allowing efficient gene transfer with small volumes of virus.

[0158] Adeno-associated virus vectors

[0159] Recombinant adeno-associated viruses (rAAV) are derived from nonpathogenic parvoviruses, evoke essentially no cellular immune response, and produce transgene expression lasting months in most systems. Moreover, like adenovirus, adeno-associated virus vectors also have the capability to infect replicating and nonreplicating cells and are believed to be nonpathogenic to humans.

[0160] AAV vectors include but are not limited to AAV1, AAV2, AAV5, AAV7, AAV8, AAV9 or AAVrh.10.

[0161] Plasmid DNA vectors

[0162] Plasmid DNA is often referred to as "naked DNA" to indicate the absence of a more elaborate packaging system. Direct injection of plasmid DNA to myocardial cells in vivo has been accomplished. Plasmid-based vectors are relatively nonimmunogenic and nonpathogenic, with the potential to stably integrate in the cellular genome, resulting in longterm gene expression in postmitotic cells in vivo. Plasmid DNA may be delivered to cells asG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0163] part of a macromolecular complex, e.g., a liposome or DNA-protein complex, and delivery may be enhanced using techniques including electroporation.

[0164] Exemplary Gene Products for Modification

[0165] Once modified gene products with a desirable activity are identified, the genome of other cells may be modified so that the modified gene product is expressed in those cells. For example, modifications of any of the gene products disclosed herein including those exemplified in the Drawings and Tables, that result in altered activity, e.g., in immune cells, may be prepared and employed in methods including methods of treatment.

[0166] Formulations and Dosages

[0167] The modified immune cells can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

[0168] In one embodiment, the immune cells may be administered by infusion or injection. Solutions of the immune cells can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0169] The pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.Sterile injectable solutions are prepared by incorporating the active agent in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and the freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile- filtered solutions.

[0170] Useful solid carriers may include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water- alcohol / gly col blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as antimicrobial agents can be added to optimize the properties for a given use. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0171] Useful dosages of the cells may be from 1 x 104to 1 x 106, 1 x 105to 1 x 107, 1 x 106to 1 x 10s, 1 x 107to 1 x 109, 1 x 108to 1 x IO10, 1 x IO10to 1 x 1012, or 1 x 1011to 1 x 1015cells.

[0172] The amount of for use alone or with other agents will vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0173] EXAMPLE

[0174] The disclosure can be better understood by reference to the following example which is offered by way of illustration. The disclosure is not limited to the examples given herein.

[0175] Methods

[0176] In vitro BE screen methods (abundance and IL2RA)

[0177] CD4+T cells were isolated from fresh peripheral blood Leukopaks (70500, STEMCELL Technologies) from healthy human donors with institutional review board-approved informed written consent (STEMCELL Technologies). The contents of the Leukopaks were washed twice with a IX volume of EasySep buffer (DPBS, 2% FBS and 1 mM EDTA (pH 8.0)) using centrifugation. The washed cells were resuspended at 200 x 106cells per millililrc in EasySep buffer and isolated with the EasySep Human CD4+T Cell Isolation Kit (STEMCELL Technologies), according to the manufacturer’s protocol. Cells were seeded at 1 * 106cells per millilitre in RPML1640 supplemented with 10% FCS, 2mM 1-glutamine(25030081. Fisher Scientific), 10 mM HEPES (H0887-100ML, Sigma), IX MEM non-essential amino acids (11140050. Fisher), 1 mM sodium pyruvate (11360070, Fisher Scientific), 100 U ml-1penicillin-streptomycin (P4333-100ML, Sigma) and 50 U ml-1IL-2 (10101641, AmeriSource Bergen). Cells were then stimulated with ImmunoCult human CD3 / CD28 / CD2 T cell activator (10990, STEMCELL Technologies) at 6.25 pl ml"1. Cells were cultured at 37 °C with 5% CO2. Following activation and electroporation, cells were split 1 :2 every 48 h to maintain an approximate density of 1 x 106cells per millilitre.

[0178] Base editor screens were performed as described in Schmidt and Ward et al. Nature 2024, including lentivirus preparation, with the following parameters. 24 hours after isolation and stimulation of the cells, ABE NGG vims was added at 2% v / v. sgRNA vims was added 24 hours after ABE at an MOI of 0.7. 24 hours later, blasticidin was added to the culture at 20 pg ml-1and puromycin at 2 pg ml-1.

[0179] Cell sorting was performed 12 days following isolation for the resting screens and 13 days following isolation for the stimulated screens. For the stimulated Tcff screen, cells were restimulated with ImmunoCult human CD3 / CD28 / CD2 T cell activator (10990, STEMCELL Technologies) 72 h before sorting, at the time of peak IL2RA expression. Before sorting, cells were counted, washed once with EasySep buffer and stained with Alexa Fluor 647 anti-human IL2RA antibody (302618, BioLegend; diluted 1:25). Cells were then washed and resuspended in EasySep buffer. During sorting, cells were gated on the top and bottom 20% of IL2RA-expressing cells were sorted into 15-ml conical tubes coated with FCS. For the abundance screens, samples were collected and lysed at the same timepoints as IL2RA screen samples were sorted. Isolated cells were pelleted, counted and lysed. Genomic DNA extraction was performed using phenol-chloroform extractions, and sgRNA libraries were amplified and prepared for sequencing using custom primers. Libraries were sequenced on an Illumina NextSeq 2000. All pooled screens were analyzed with MAGeCK42 (v0.5.9.5) comparing the IL2RA high bin cells to IL2RA low bin from the same condition, or the expanded cells to the plasmid guide distribution.

[0180] In vivo BE screen methods (IFNy and tumor infiltration)

[0181] NSG mice were first injected with 4 million anti-CD3 scFv expressing A375 tumor cells (using the clone described in benchmarking experiments above) into the unilateral flank. The isolation of primary human T cells from human PBMCs was conducted via negative selection using the EasySep Human T Cell Isolation Kit following the manufacturer’s instructions (STEMCELL). Leukopaks from two deidentified healthy donors (STEMCELL) were used for this purpose and came with consent from donors and protocols approved by anInstitutional Review Board. After isolation, T cells were activated using CTS Dynabeads Human T-Cell Activator CD3 / CD28 and cultured at a density of lx 106 cells / ml in complete human T cell growth medium defined as X-Vivo-15 media (Lonza) supplemented with 5% human serum (Gemini), 50 pM 2-mercaptoethanol (Thermo Fisher), and 10 mM N-acetyl-L-cystcinc (MilliporcSigma). T cells were split every 48-72 hours. Base editing and transduction of T cells was performed as described above for in vitro screens.

[0182] 11 days after the NSG mice were injected with the tumor cells, mice were injected with 4 million T cells per NSG mouse and monitored for tumor size in the subsequent days. At 4 million T cells per tumor, 18 mice sufficed to achieve 1000X library coverage at the time of T cell injection. 11 days after T cell injection, the mice were sacrificed for collection of the tumors from all mice. Tumors were digested and T cells isolated using density gradient centrifugation (Ficoll). After isolation, T cells for each donor were pooled from all tumors. Once T cells were pooled and genomic DNA was isolated from the cells using the NucleoSpin Blood XL kit (Machcrcy-Nagcl). Genomic DNA concentration was quantified using Invitrogcn IX dsDNA High Sensitivity assay kit (Thermo Fisher) on a Qubit Fluorometer (Invitrogen). PGR of the amplicon containing the guide sequences was carried out using Ex Taq DNA Polymerase (Takara Bio) and P5 / P7 primers (IDT). The resulting amplicons were purified using SPRIselect Beads (Beckman-Coulter) and QC was conducted using a DI 000 ScreenTape assay on a TapeStation (Agilent) prior to next-generation sequencing. Samples were pooled and sequenced on NextSeq 2000. MAGeCK52 v0.5.9.5 was used to quantify guide counts in each sample and tested for guide enrichment. Samples of T cells collected before infusion into mice (input) were compared to samples collected from tumors. Guides with a read count of < 50 in the control samples were filtered out. In an additional cohort, we performed a second screen, where at the final timepoint we sorted the T cells from the tumors based on level of interferon production. In this case, after isolation from tumors, the T cells were stimulated and sorted based on gating for the top and bottom 20% of IFN-g levels by flow cytometry. After sorting, these T cells were processed as described for the prior screens. Thus, we performed screens based on abundance and persistence relying on the sgRNA frequencies detected in tumors, as well as screens based on a marker of effector function (interferon gamma production by TIL).

[0183] Nascent RNA Screens

[0184] Nascent RNA screens were performed with cells labeled using the Click-iT™ RNA Alexa Fluor™ 488 Imaging Kit. CD4+ T cells were incubated with 5-ethynyl uridine (EU) for one hour prior to fixation and staining.

[0185] ResultsG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0186] Figure 1 describes a population of T cells contacted with a library of sgRNAs targeting coding regions within genes of the RNA Pol II pre-initiation complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. Following base editing, the T cells were screened for IFNy production by sorting cells into high and low IFNy expression bins, and the effect of each sgRNA-mcdiatcd edit is determined by calculating the log2FoldChange between the IFNy high bin and IFNy low bin. The specific edits are identified by the gene name and the predicted amino acid change, enabling the selection of T cells with enhanced or suppressed IFNy production for therapeutic applications.

[0187] Figure 2 describes a population of T cells contacted with a library of sgRNAs targeting coding regions within genes of the Mediator complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. After base editing, the T cells were screened for IFNy production by sorting cells into high and low IFNy expression bins, and the effect size of each sgRNA-mediated edit is determined by calculating the log2FoldChange between the IFNy high bin and IFNy low bin. The specific edits arc identified by the gene name and the predicted amino acid change, allowing for the selection of T cells with enhanced or suppressed IFNy production for therapeutic use.

[0188] Figure 3 describes a population of T cells contacted with a library of sgRNAs targeting coding regions within genes of the RNA Pol II pre-initiation complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. Following base editing, the T cells are screened for tumor infiltration by comparing the abundance of each sgRNA-modified T cell in tumor tissue versus the input population, with the effect size represented as the log2FoldChange of sgRNA frequency in tumor relative to input. The specific edits are identified by the gene name and the predicted amino acid change, enabling the selection of T cells with enhanced tumor infiltration for therapeutic applications.

[0189] Figure 4 describes a population of stimulated CD4+ T cells contacted with a library of sgRNAs targeting coding regions within genes of the RNA Pol II pre-initiation complex or Mediator complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. After base editing, the CD4+ T cells are screened for IL2RA expression by sorting cells into high and low IL2RA expression bins, and the effect size of each sgRNA-mediated edit is determined by calculating the log2FoldChange between the IL2RA high bin and IL2RA low bin. The specific edits are identified by the gene name and the predicted amino acid change, allowing for the selection of CD4+ T cells with enhanced IL2RA expression for therapeutic use.G L2025-001 / / SF2O25-143 / / 3730.239WO1

[0190] Figure 5 provides a population of resting CD4+ T cells contacted with a library of sgRNAs targeting coding regions within genes of the RNA Pol II pre-initiation complex or Mediator complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. After base editing, the CD4+ T cells are screened for IL2RA expression by sorting cells into high and low IL2RA expression bins, and the effect size of each sgRNA-mediated edit is determined by calculating the log2FoldChange between the IL2RA high bin and IL2RA low bin. The specific edits are identified by the gene name and the predicted amino acid change, enabling the selection of resting CD4+ T cells with enhanced IL2RA expression for therapeutic applications.

[0191] Figure 6 provides human CD4+ T cells individually contacted with specific sgRNAs targeting coding regions within genes of the RNA Pol II pre-initiation complex or Mediator complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. Following base editing and stimulation, IL2RA expression is validated in an arrayed format using flow cytometry, with each sgRNA represented as a separate bar and the time after stimulation displayed on the y axis. The predicted edits are indicated on the x axis, and the log2Fold Change in IL2RA expression is calculated relative to AAVS1 edit controls, enabling identification and selection of CD4+ T cells with enhanced IL2RA expression for therapeutic use.

[0192] Figure 7 provides human CD4+ T cells individually contacted with specific sgRNAs targeting coding regions within genes of the RNA Pol II pre-initiation complex or Mediator complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. Following base editing and stimulation, IL2RA screen hits are validated in an arrayed format by measuring surface protein expression using flow cytometry, with mean fluorescence intensity (MFI) assessed for each sgRNA-modified cell. A strong positive correlation is observed between MFI and the screen Log2Fold Change, enabling identification and selection of CD4+ T cells with enhanced IL2RA surface expression for therapeutic use.

[0193] Figure 8 provides stimulated CD4+ T cells contacted with a library of sgRNAs targeting coding regions within genes of the RNA Pol II pre-initiation complex or Mediator complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. After base editing, the cells are screened in vitro for nascent RNA production by sorting cells into high and low nascent RNA expression bins, with effect size represented as the mean log2FoldChange between the EU high bin and EU low bin. The specific edits are identified by the gene name and predicted amino acid change, with amino acid numbering shifted by 1 relative to the provided tables / figures so that the first amino acid is Ml V. Select EU screen hitsG L2025-001 / / SF2O25-143 / / 3730.239WO1

[0194] are mapped onto published protein structures, highlighting that disruption of CCNC increases nascent RNA production. Validation of these nascent RNA screen hits is performed using flow cytometry, confirming that mutations in CCNC increase nascent RNA production in stimulated CD4+ T cells, with amino acid numbering shifted accordingly.

[0195] Figure 9 provides stimulated CD4+ T cells contacted with a library of sgRNAs targeting coding regions within genes of the RNA Pol II pre-initiation complex or Mediator complex, together with a base editor such as an adenine or cytosine base editor delivered via a viral vector. After base editing, the cells are screened in vitro for IL2RA expression and cell abundance by sorting cells into high and low IL2RA expression bins, with effect size represented as the mean log2FoldChange between the IL2RA high bin and IL2RA low bin. The specific edits are identified by the gene name and predicted amino acid change, with amino acid numbering shifted by 1 relative to the provided tables / figures so that the first amino acid is M1V. Select stimulated IL2RA screen hits are mapped onto published protein structures, highlighting that disruption of external residues on GTF2A1 decreases IL2RA expression without decreasing cell abundance. Additionally, select residues on MED 24 and MED 16 are depicted as increasing IL2RA expression on CD4+ T cells, with amino acid numbering shifted accordingly.

[0196] Figure 11 CD 19 CAR T cells base edited at the POL2A_5093 site using a specific sgRNA and a base editor delivered via a viral vector, resulting in modified CAR T cells with enhanced anti-tumor activity. These POL2A_5093 -edited CAR T cells were administered to NSG mice bearing CD19-A375 melanoma tumors, leading to reduced tumor growth compared to control-treated mice. The edited CAR T cells demonstrated increased infiltration into tumor tissue, as measured by the percentage of CAR T cells within tumors, and also show elevated levels of human CAR T cells among total CD451cells in peripheral blood following tumor clearance, indicating improved persistence and therapeutic efficacy.

[0197] Figure 12 provides CD 19 CAR T cells base edited at the TAF9_2303 site using a specific sgRNA and a base editor delivered via a viral vector, resulting in modified CAR T cells with enhanced anti-tumor activity. These TAF9_2303-edited CAR T cells were administered to NSG mice bearing CD19-A375 melanoma tumors, leading to reduced tumor growth compared to control-treated mice. The edited CAR T cells exhibit increased infiltration into tumor tissue, as measured by the percentage of CAR T cells within tumors, demonstrating improved therapeutic efficacy.

[0198] Figure 13 provides CD 19 CAR T cells base edited at the POL2A_5698 and / or TAF8_2254 sites using specific sgRNAs and a base editor delivered via a viral vector, resultingin modified CAR T cells with potentially enhanced anti-tumor activity. These POL2A_5698-or TAF8_2254-edited CAR T cells were administered to NSG mice bearing CD19-A375 melanoma tumors, leading to reduced tumor growth compared to control-treated mice, indicating improved therapeutic efficacy of the base-edited CAR T cells.

[0199] Figures 14-16 provide lists in which T cells arc contacted with sgRNAs selected from Figures 14-16, which specify the target genes, accession numbers, protein sequences, and chromosomal locations, along with a base editor such as an adenine or cytosine base editor delivered via a viral vector. The sgRNAs induce specific edits at defined amino acid positions, with the starting amino acid numbered as 0, and the resulting effect on T cell function is assessed. The edits and their corresponding effects, as detailed in FIGS. 14-16, enable the selection of T cells with desired functional attributes, such as enhanced proliferation, cytokine production, or altered immune activity, for therapeutic applications.

[0200] Bibliography

[0201] Schmidt, R., Ward, C.C., Dajani, R. et al. Base-editing mutagenesis maps alleles to tunc human T cell functions. Nature 625, 805-812 (2024). https: / / doi.org / 10.1038 / s41586-023-06835-6.

[0202] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0203] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.

[0204] All publications, patents, and patent applications, Genbank sequences, websites and other published materials referred to throughout the disclosure herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application, Genbank sequences, websites and other published materials was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts with a term defined herein, this specification shall control.

Claims

WHAT IS CLAIMED IS:

1. A method comprising:contacting T cells with one or more sgRNAs of Figure 15 targeted to one or more coding regions in one or more genes of PIC or Mediator Complex and a base editor or nucleic acid encoding the base editor; andselecting one or more T cells that have enhanced T cell function or suppressed T cell function and optionally sorting said T cells.

2. The method of claim 1, wherein the T cells are contacted with a library of sgRNAs targeted to the one or more coding regions in a plurality of PIC or Mediator Complex genes.

3. The method of claim 1, wherein a library of viruses expresses the one or more sgRNAs.

4. The method of claim 1, wherein a virus encodes the base editor.

5. The method of claim 1, wherein the sgRNA and the nucleic acid encoding the base editor are on different nucleic acid molecules.

6. The method of claim 3, wherein the virus is a lentivirus, retrovirus, adenovirus, herpesvirus or adeno-associated virus.

7. The method of claim 1, wherein the base editor comprises ABE.

8. The method of claim 1, wherein the base editor comprises CBE.

9. The method of claim 1, wherein the cells are selected for enhanced T cell proliferation, IL2RA expression, cytokine production, increased tumor infiltration, increased or decreased IFN-y production or any combination thereof or suppressed immune function.

10. The method of claim 1, wherein the cells are sorted to separate cells that have enhanced T cell proliferation, IL2RA expression, cytokine production, increased tumor infiltration, increased or decreased IFN-y production or any combination thereof or suppressed immune function.

11. The method of any one of claims 1 to 10, wherein the sgRNAs comprise one or more of the sequences in Figure 14 or a sequence with at least 85% nucleic acid sequence identity thereto.

12. The method of any one of claims 1 to 11, wherein the one or more coding regions that are altered are in one or more of the genes in Figures 14-16 or Figure 10 or are modified at a site in Figures 14-16, Figure 10 or a sequence with at least 85% nucleic acid sequence identity thereto.

13. The method of any one of claims 1 to 12, wherein the sgRNAs comprise one or more of SEQ ID NOs: 1-20.

14. A population of cells obtained by the method of any one of claims 1 to 13.

15. An isolated human cell obtained by the method of any one of claims 1 to 13.

16. An isolated cell having a base edited gene in Figures 14-16 which alters one or more activities of the cell.

17. The isolated cell of claim 16 which has enhanced T cell proliferation, IL2RA expression, cytokine production relative to a corresponding cell that lacks the base edited gene.

18. The isolated cell of any one of claims 15 to 16 which is modified at a site in Figures 14-16 or a sequence with at least 85% nucleic acid sequence identity thereto.

19. The isolated cell of any one of claims 15 to 18 which has a modified gene selected from MED24, MED 16, TBP, GTF2A1, TAF3, ERCC3, GTF2F1, GTF2F2, CCNC, POLR2A, TAF9, TAF8, TAF3 or combination thereof.

20. The isolated cell of claim 19, wherein the gene modification / edit are selected from those provided in Figure 10.

21. A method to prevent, inhibit or treat cancer in a mammal comprising administering to the mammal a composition having a plurality of the isolated cells of any one of claims 15 to 20.

22. The method of claim 21, wherein the cells arc systemically administered.

23. The method of claim 21 or 22, wherein the mammal is a human.

24. The method of any one of claims 21 to 23, wherein the cancer is an immune cell cancer.

25. The method of claim 24, wherein the cancer is sarcoma, lymphoma, melanoma, or carcinoma, or is lung cancer, breast cancer, prostate cancer, pancreatic cancer or ovarian cancer.

26. The method of any one of claims 21 to 25, wherein the cell is a CAR-T cell.

27. The method of any one of claims 21 to 26, wherein the cells are autologous cells.

28. The method of any one of claims 21 to 26, wherein the cells are allogeneic cells.

29. A method to prevent, inhibit or treat an autoimmune disease in a mammal, comprising administering to the mammal a composition having a plurality of the isolated cell of any one of claims 15 to 18.

30. The method of claim 29, where the cells are systemically administered.

31. The method of claim 29 or 30, wherein the mammal is a human.

32. The method of any one of claims 29 to 31, wherein the autoimmune disease is multiple sclerosis.

33. The method of any one of claims 29 to 32, wherein the cells are autologous cells.

34. The method of any one of claims 29 to 32, wherein the cells are allogeneic cells.

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