Compositions and methods for genetically modifying transforming growth factor beta receptor type 2 (tgfp2)

Abstract

Compositions and methods for editing, e.g., altering, DNA sequences within TGFβR2 are provided. Compositions and methods for reducing or eliminating TGFβR2 protein expression in a cell are provided. Compositions and methods for immunotherapy are provided.

Description

I. Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 519,503, filed August 14, 2023, and U.S. Provisional Application No. 63 / 610,602, filed December 15, 2023, pursuant to 35 USC 119(e), the contents of each of which are incorporated herein by reference in their entirety.

[0002] II. Reference to Electronic Sequence Lists This application contains a sequence list submitted electronically in XML file format and hereby incorporated in its entirety by reference. The XML file was created on August 5, 2024, named "01155-0065-00PCT.xml", and has a size of 2,227,486 bytes. Summary of the Invention

[0003] This disclosure specifically relates to gene modification of the transforming growth factor β receptor type 2 (TGFβR2) gene. This disclosure also relates to the CRISPR / Cas9 genome editing system.

[0004] Transforming growth factor β (TGF-β) is a secreted cytokine with pleiotropic effects on processes ranging from development and oncogenesis to immune signaling. Within the immune system, TGF-β signaling maintains homeostasis by promoting self-tolerance and suppressing inflammation. It achieves this through a wide range of mechanisms, including disrupting the antigen-presenting capacity of dendritic cells; mitigating the cytotoxicity of natural killer cells; inhibiting macrophages from acquiring a pro-inflammatory phenotype; and suppressing T cell activation, differentiation, and proliferation (Batlle and Massague 2019).

[0005] The interaction between TGF-β signaling and T cell biology has attracted considerable interest, primarily due to its role in cancer pathophysiology and its significance for developing novel therapies. While TGF-β typically functions as a tumor suppressor, inhibiting the proliferation of precancerous cells and inducing apoptosis, many cancers possess mutations that inactivate the TGF-β pathway, allowing them to evade the antitumor effects of TGF-β signaling. Paradoxically, in TGF-β-resistant cases, high levels of TGF-β may promote tumorigenesis by protecting cancer cells from immune system attack. For example, high levels of TGF-β within the tumor microenvironment may promote T cell rejection from the tumor (Tauriello et al., 2018). Furthermore, TGF-β signaling may prevent primary T cells from differentiating into helper T cells, thereby reducing immune surveillance within the tumor microenvironment (Sad and Mosmann, 1994). As a final example, TGF-β may also inhibit T cell proliferation in response to tumors (Donkor et al., 2011).

[0006] Therefore, there is a need for improved methods and compositions for modifying cells to overcome TGF-β-mediated immunosuppression. In particular, there is an unmet need for methods to mitigate the inhibitory effects of TGF-β signaling on T cell activation, differentiation, and proliferation.

[0007] This article provides compositions and methods for genetically modifying the TGFβR2 sequence, as well as cells with genetic modifications in the TGFβR2 sequence and their uses in various methods, such as promoting immune responses, for example in immuno-oncology and infectious diseases. Methods for using the provided compositions to promote immune responses and treat cancer or infectious diseases are also provided.

[0008] This disclosure relates to cell populations comprising cells with genetic modifications in the TGFβR2 sequence as provided herein. These cells can be used in adoptive T-cell transfer therapy.

[0009] This disclosure relates to compositions and uses of genetically modified cells having the TGFβR2 sequence, which are used in therapies such as cancer therapy and immunotherapy. This disclosure relates to and provides gRNA molecules, CRISPR systems, cells, and methods for use in cellular genome editing.

[0010] This article provides an engineered cell that contains gene modifications in the TGFβR2 sequence within the genomic coordinates chr3:0606891-30691605.

[0011] In some embodiments, this disclosure provides engineered cells in which the surface expression of TGFβR2 protein is reduced or eliminated due to genetic modifications in the TGFβR2 gene. Engineered cell compositions produced by the methods disclosed herein have desired properties, including, for example, reduced or eliminated TGFβR2 protein expression, reduced chronic TGFβR2-mediated aberrant immune responses (such as T cell exhaustion), thereby enhancing immune responses.

[0012] The use of any of the aforementioned embodiments of the cell composition or formulation for preparing a medicament for treating a subject is also disclosed. The subject may be a human or an animal (e.g., a human or a non-human animal, such as a cynomolgus monkey). Preferably, the subject is a human.

[0013] Also disclosed are any of the aforementioned compositions or formulations for gene modifications (e.g., insertions, substitutions, or deletions) to generate the TGFβR2 gene sequence. In some embodiments, the intra-sequence gene modification results in a change in the nucleic acid sequence that prevents the translation of the full-length protein prior to the gene modification at the genomic locus, for example, by forming frameshift or nonsense mutations, causing premature termination of translation. Gene modifications may include insertions, substitutions, or deletions at splice sites (i.e., splice acceptor sites or splice donor sites), such that aberrant splicing results in frameshift, nonsense, or truncated mRNA, thereby causing premature termination of translation. Gene modifications may also disrupt the translation or folding of the protein encoding the gene, leading to premature termination of translation.

[0014] In another aspect, this disclosure provides a method for treating a subject, the method comprising administering cells (e.g., a cell population) prepared by a cell preparation method described herein, such as any of the foregoing aspects and embodiments of the cell preparation method.

[0015] Also disclosed are any of the aforementioned compositions or preparations for generating gene modifications (e.g., insertions, substitutions, or deletions) in the TGFβR2 sequence. In some embodiments, the gene modification within the sequence results in a change in the nucleic acid sequence that prevents the translation of the full-length protein prior to the gene modification at the genomic locus, for example, by forming frameshift or nonsense mutations, causing premature termination of translation. Gene modifications may include insertions, substitutions, or deletions at splice sites (i.e., splice acceptor sites or splice donor sites), such that aberrant splicing results in frameshift, nonsense, or truncated mRNA, thereby causing premature termination of translation. Gene modifications may also disrupt the translation or folding of the protein encoding the protein, leading to premature termination of translation.

[0016] In another aspect, this disclosure provides a method for providing enhanced immunotherapy to a subject, the method comprising administering to the subject an effective amount of cells as described herein, such as cells of any of the foregoing cell aspects and embodiments.

[0017] In some embodiments, the engineered cells contain gene modifications within any of the genomic coordinates listed in Table 2. In some embodiments, the gene modifications are within genomic coordinates targeted by a guide RNA containing a guide sequence of any of SEQ ID NO: 1-49.

[0018] Further embodiments are provided throughout and described in the claims and drawings. Attached Figure Description

[0019] Figure 1 The average insertion / deletion percentage and average pSMAD2 / 3 negative T cells are shown after editing TGFBR2 with the Nme2Cas9 base editor.

[0020] Figure 2A The average insertion / deletion percentage in T cells is shown after editing TGFBR2 with the Nme2Cas9 base editor at various doses.

[0021] Figure 2B The average pSMAD2 / 3 negative T cells were shown after TGFBR2 was edited with the Nme2Cas9 base editor at various doses.

[0022] Figure 3 The percentage of allogeneic CD70 CAR-T cell editing per edit is shown for each of the three donors, as assessed by flow cytometry or genome sequencing (results from each donor are shown as solid dots). Detailed Implementation

[0023] Reference will now be made in detail to certain embodiments of this disclosure, examples of which are illustrated in the accompanying drawings. While this doctrine has been described in conjunction with various embodiments, it is not intended to limit this doctrine to those embodiments. Rather, as those skilled in the art will understand, this doctrine encompasses various alternatives, modifications, and equivalents.

[0024] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. In the event of any material incorporated by reference that contradicts any terminology defined herein or any other express content herein, this specification shall prevail.

[0025] This document provides the following numbered implementation schemes: Implementation scheme 1 is an engineered cell containing gene modifications within the genomic coordinates chr3:30606891-30691605.

[0026] Implementation scheme 2 is an engineered cell that has reduced or eliminated surface expression of TGFβR2 protein compared to unmodified cells, and contains gene modifications within the genomic coordinates chr3:30606891-30691605.

[0027] Implementation scheme 3 is an engineered cell as described in implementation scheme 1 or 2, wherein the gene modification is within genomic coordinates targeted by a guide RNA, the guide RNA comprising a guide sequence of any of SEQ ID NO: 1-49.

[0028] Implementation scheme 4 is an engineered cell as described in any one of implementation schemes 1-3, wherein the engineered cell has reduced or eliminated TGFβR2 surface expression relative to unmodified cells, and contains gene modifications within any of the genomic coordinates listed in Table 2.

[0029] Implementation scheme 5 is an engineered cell as described in any one of implementation schemes 1-4, wherein the gene modification is within the genomic coordinates selected from the following: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; chr3:30672133-30672157; chr3:30606891-30606915; chr3:30606892-30606916; chr3:30606896-30606920; chr3:30606897-30606897-30606898-30606920; chr3:30606897-30606898-3060689 ... 0606921;chr3:30606898-30606922;chr3:30606899-30606923;chr3:306 06908-30606932;chr3:30606909-30606933;chr3:30606910-30606934;c hr3:30606917-30606941; chr3:30606958-30606982; chr3:30606959-306 06983;chr3:30606960-30606984;chr3:30606964-30606988;chr3:30606 965-30606989;chr3:30644900-30644924;chr3:30671667-30671691;chr 3:30671670-30671694; chr3:30671753-30671777; chr3:30671762-30671 786;chr3:30671766-30671790;chr3:30672034-30672058;chr3:3067212 6-30672150;chr3:30672128-30672152;chr3:30672131-30672155;chr3: 30672135-30672159;chr3:30672139-30672163;chr3:30672140-3067216 4;chr3:30672140-30672164;chr3:30672141-30672165;chr3:30672190- 30672214;chr3:30672204-30672228;chr3:30672432-30672456;chr3:30 672433-30672457;chr3:30672434-30672458;chr3:30674211-30674235;chr3:30688450-30688474; chr3:30688459-30688483; chr3:30688460-30688484; chr3:30688476-30688500; chr3:30688478-30688502; chr3:30688479-30688503; chr3:30691436-30691460; and chr3:30691581-30691605.

[0030] Embodiment 6 is an engineered cell as described in any one of Embodiments 1-5, wherein the gene modification is within genomic coordinates targeted by a guide RNA, the guide RNA comprising a guide sequence of any one of SEQ ID NO: 1-5.

[0031] Implementation scheme 7 is an engineered cell as described in any one of implementation schemes 1-6, wherein the gene modification is within the genomic coordinates selected from the following: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; and chr3:30672133-30672157.

[0032] Embodiment 8 is an engineered cell as described in any one of Embodiments 1-7, wherein the gene modification is within genomic coordinates targeted by a guide RNA, the guide RNA comprising the guide sequence of SEQ ID NO: 1.

[0033] Implementation scheme 9 is an engineered cell as described in any one of implementation schemes 1-8, wherein the gene modification is located within the genomic coordinates chr3:30674205-30674229.

[0034] Embodiment 10 is a composition comprising a guide RNA and optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the guide RNA comprises (a) a guide sequence selected from SEQ ID NO: 1-49; (b) a guide sequence of at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides selected from SEQ ID NO: 1-49; (c) a guide sequence having at least 85%, 90%, or 95% identity with a sequence selected from SEQ ID NO: 1-49; (d) a sequence comprising 10 consecutive nucleotides ± 10 nucleotides of genomic coordinates listed in Table 2; (e) at least 20, 21, 22, 23, or 24 consecutive nucleotides from the sequence of (d); and (f) a guide sequence having at least 85%, 90%, or 95% identity with a sequence selected from (d).

[0035] Embodiment 11 is a composition as described in Embodiment 10, said composition being used to alter the DNA sequence within the TGFβR2 gene in cells.

[0036] Embodiment 12 is a pharmaceutical composition comprising the composition described in Embodiment 10, or the use of the composition described in Embodiment 10 for inducing double-strand breaks or single-strand breaks in the TGFβR2 gene in cells, altering the nucleic acid sequence of the TGFβR2 gene in cells, or reducing the expression of the TGFβR2 gene in cells.

[0037] Embodiment 13 is a method for manufacturing engineered human cells having reduced or eliminated surface expression of TGFβR2 protein compared to unmodified cells, the method comprising contacting the cells with a composition as described in Embodiment 10.

[0038] Implementation Scheme 14 is a method for reducing the surface expression of TGFβR2 protein in cells relative to unmodified cells, the method comprising contacting cells with a composition comprising a guide RNA and optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the guide RNA comprises a guide sequence selected from SEQ ID NO: 1-49; b. a guide sequence of at least 20, 21, 22, 23, 24 or 25 consecutive nucleotides selected from SEQ ID NO: 1-49; c. a guide sequence having at least 85%, 90% or 95% identity with a sequence selected from SEQ ID NO: 1-49; d. a sequence comprising 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; e. a sequence of at least 20, 21, 22, 23 or 24 or 25 consecutive nucleotides from (d); and f. a guide sequence having at least 85%, 90% or 95% identity with a sequence selected from (d).

[0039] Embodiment 15 is a composition, use, or method as described in any one of Embodiments 10-14, wherein the guide RNA comprises the guide sequence of SEQ ID NO: 1.

[0040] Embodiment 16 is a composition, use, or method as described in any one of Embodiments 10-15, wherein the RNA-guided DNA binder is a base editor.

[0041] Embodiment 17 is a cell population comprising engineered cells produced by using a composition as described in any one of Embodiments 10-12, 15 and 16 or a method as described in any one of Embodiments 13-16.

[0042] Embodiment 18 is a pharmaceutical composition comprising (a) engineered cells produced by a composition or method as described in any one of Embodiments 10-16; or (b) a cell population as described in Embodiment 17.

[0043] Embodiment 19 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-18, wherein the genetic modification comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates.

[0044] Embodiment 20 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-19, wherein the genetic modification comprises at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates.

[0045] Embodiment 21 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-20, wherein the gene modification comprises insertion, deletion, or substitution.

[0046] Embodiment 22 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-21, wherein the gene modification comprises an insertion / deletion, C-to-T substitution, or A-to-G substitution within the genomic coordinates.

[0047] Embodiment 23 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-22, wherein the gene modification comprises an insertion / deletion.

[0048] Embodiment 24 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-23, wherein the gene modification comprises the insertion of a heterologous coding sequence.

[0049] Embodiment 25 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-24, wherein the gene modification comprises substitution.

[0050] Embodiment 26 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-25, wherein the genetic modification comprises A to G substitutions.

[0051] Embodiment 27 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-26, wherein the gene modification comprises C to T substitution.

[0052] Embodiment 28 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-27, wherein the cell is engineered using a genome editing system.

[0053] Embodiment 29 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 28, wherein the genome editing system comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0054] Embodiment 30 is the composition as described in Embodiment 29, wherein the nucleic acid encoding the RNA-guided DNA binder comprises mRNA, and the mRNA comprises an open reading frame (ORF) encoding the RNA-guided DNA binder.

[0055] Embodiment 31 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 29 or 30, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid comprises a Cas9 nuclease.

[0056] Embodiment 32 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 29-31, wherein the RNA-guided DNA binder is a nuclease.

[0057] Embodiment 33 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 29-32, wherein the RNA-guided DNA binder is a Cas9 nuclease.

[0058] Implementation scheme 34 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 33, wherein the Cas9 is NmeCas9.

[0059] Embodiment 35 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 34, wherein NmeCas9 comprises an amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 832-834 and an ORF encoding NmeCas9 having at least 90% identity with a sequence selected from SEQ ID NO: 832-834.

[0060] Embodiment 36 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 35, wherein the ORF encoding the amino acid sequence has at least 85% identity with a sequence selected from SEQ ID NO: 802, 803, and 805-807.

[0061] Embodiment 37 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 31-36, wherein the nuclease has double-stranded endonuclease activity.

[0062] Embodiment 38 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 31-36, wherein the nuclease has nicking enzyme activity.

[0063] Embodiment 39 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 31-36, wherein the nuclease is non-catalytically active.

[0064] Implementation scheme 40 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of implementation schemes 31-36, wherein the nuclease further comprises a heterologous functional domain.

[0065] Embodiment 41 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 40, wherein the nuclease is a nicking enzyme and the heterologous functional domain is a deaminase.

[0066] Embodiment 42 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 41, wherein the deaminase is cytidine deaminase or adenine deaminase.

[0067] Implementation scheme 43 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 42, wherein the deaminase is cytidine deaminase.

[0068] Embodiment 44 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 43, wherein the deaminase is an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.

[0069] Embodiment 45 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 41-44, wherein the nuclease and the deaminase comprise an amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 831, 835-838 and an ORF encoding an amino acid sequence having at least 90% identity with SEQ ID NO: 831.

[0070] Embodiment 46 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 45, wherein the ORF encoding the amino acid sequence has at least 85% identity with SEQ ID NO: 801.

[0071] Embodiment 47 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 43-46, wherein the engineered cell, cell population, pharmaceutical composition, or method further comprises a uracil glycosidase inhibitor (UGI) or a nucleic acid encoding UGI, wherein the nuclease does not contain UGI, or the nucleic acid encoding the nuclease does not encode UGI.

[0072] Embodiment 48 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 47, wherein the UGI comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 859, or an ORF encoding an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 859.

[0073] Embodiment 49 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 48, wherein the ORF encoding the amino acid sequence has at least 85% identity with any one of SEQ ID NO: 823-826, optionally SEQ ID NO: 823.

[0074] Embodiment 50 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 45-49, wherein the ORF is a modified ORF.

[0075] Embodiment 51 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 45-50, wherein the nuclease has nicking enzyme activity.

[0076] Embodiment 52 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiments 45-51, wherein the nuclease or the nuclease encoded by the nucleic acid comprises Neisseria meningitidis (… N. meningitidis Cas9 (NmeCas9).

[0077] Embodiment 53 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 52, wherein NmeCas9 comprises Nme2Cas9.

[0078] Embodiment 54 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 52 or 53, wherein the nucleic acid encoding Nme2Cas9 is mRNA, and the mRNA comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity with SEQ ID NO: 834.

[0079] Embodiment 55 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 45-54, wherein the nucleic acid encoding the base editor comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% identity with SEQ ID NO: 801.

[0080] Embodiment 56 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 3-55, wherein the guide RNA is a dual guide RNA (dgRNA).

[0081] Embodiment 57 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 3-55, wherein the guide RNA is a single guide RNA (sgRNA).

[0082] Embodiment 58 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 57, wherein the sgRNA is an Nme sgRNA comprising a guide region and a conserved region.

[0083] Embodiment 59 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 58, wherein the conserved region comprises one or more of the following: (a) a shortened repeat / anti-repeat region, wherein the shortened repeat / anti-repeat region is missing 2-24 nucleotides relative to SEQ ID NO: 700, wherein (i) one or more of nucleotides 37-48 and 53-64 are missing relative to SEQ ID NO: 700 and optionally one or more of nucleotides 37-64 are substituted relative to SEQ ID NO: 700; and (ii) nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b) a shortened hairpin 1 region, wherein the shortened hairpin 1 is missing 2-10 nucleotides, optionally 2-8 nucleotides, relative to SEQ ID NO: 700, wherein (i) one or more of nucleotides 82-86 and 91-95 are missing relative to SEQ ID NO: 700 and optionally one or more of positions 82-96 are missing relative to SEQ ID NO: 700. 700 is substituted; and (ii) nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (c) a shortened hairpin 2 region, wherein the shortened hairpin 2 is missing 2-18 nucleotides relative to SEQ ID NO: 700, optionally 2-16 nucleotides, wherein (i) one or more of nucleotides 113-121 and 126-134 is missing relative to SEQ ID NO: 700 and optionally one or more of nucleotides 113-134 is substituted relative to SEQ ID NO: 700; and (ii) nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides; one or two nucleotides 144-145 are optionally missing relative to SEQ ID NO: 700; optionally, wherein at least 10 nucleotides are modified nucleotides.

[0084] Embodiment 60 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 58 or 59, wherein the conserved region comprises a modified nucleotide sequence selected from the modified conserved region Nme guide RNA motif in Table 6, and wherein the conserved region is located at the 3' of the guide region.

[0085] Embodiment 61 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 58, wherein the guide RNA comprises a nucleotide sequence selected from any of SEQ ID NO: 701-706, and wherein N represents a guide sequence of any of SEQ ID NO: 1-49.

[0086] Implementation scheme 62 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 61, wherein each nucleotide is any natural or non-natural nucleotide.

[0087] Embodiment 63 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 62, wherein the guide RNA is modified according to the sequence or modification pattern listed in Tables 6-7, wherein N is the guide sequence, N, A, C, G, and U are ribonucleotides (2'-OH), "m" indicates 2'-O-Me modification, "f" indicates 2'-fluorine modification, and "*" indicates phosphate thioester linkage between nucleotides.

[0088] Embodiment 64 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 3-63, wherein the guide RNA comprises at least one end modification.

[0089] Embodiment 65 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 64, wherein the modification includes a 5' end modification.

[0090] Embodiment 66 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 64 or 65, wherein the modification includes a 3' end modification.

[0091] Embodiment 67 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 64-66, wherein the guide RNA comprises a modification in a hairpin region.

[0092] Implementation scheme 68 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 67, wherein the modification in the hairpin region is also an end modification.

[0093] Embodiment 69 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 64-68, wherein the modification comprises a nucleotide modified with 2'-O-methyl (2'-O-Me).

[0094] Embodiment 70 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 64-69, wherein the modification comprises a phosphate thioester (PS) bond between nucleotides.

[0095] Embodiment 71 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 64-70, wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide linked to a 3' adjacent nucleotide by a phosphate thioester (PS) bond.

[0096] Embodiment 72 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 64-71, wherein the modification comprises a 2'-fluorine (2'F) modified nucleotide.

[0097] Embodiment 73 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 65-72, wherein the 5' end modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide at nucleotides 1-3 at the 5' end of the guide sequence, linked to the 3' adjacent nucleotide by a phosphate thioester (PS) bond.

[0098] Embodiment 74 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 3-73, wherein the guide RNA is associated with lipid nanoparticles (LNPs).

[0099] Embodiment 75 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 74, wherein the LNP comprises cationic lipids, auxiliary lipids, neutral lipids, occult lipids, or a combination of two or more thereof.

[0100] Embodiment 76 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 75, wherein the cationic lipid is octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadecano-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester.

[0101] Embodiment 77 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 75 or 76, wherein the auxiliary lipid is cholesterol.

[0102] Embodiment 78 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 75-77, wherein the neutral lipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC).

[0103] Embodiment 79 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 75-78, wherein the elusive lipid is 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol 2000 (PEG2k-DMG).

[0104] Embodiment 80 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 75-79, wherein the LNP comprises octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadecano-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester; DSPC; cholesterol; and PEG2k-DMG.

[0105] Embodiment 81 is a pharmaceutical composition comprising engineered cells as described in any one of Embodiments 1-80.

[0106] Embodiment 82 is a cell population comprising engineered cells as described in any one of Embodiments 1-80.

[0107] Embodiment 83 is a pharmaceutical composition comprising a cell population, wherein the cell population comprises a plurality of engineered cells as described in any one of Embodiments 1-80.

[0108] Embodiment 84 is a pharmaceutical composition as described in Embodiment 83, wherein the pharmaceutical composition further comprises a pharmaceutical excipient.

[0109] Implementation scheme 85 is a method of administering engineered cells, cell populations or pharmaceutical compositions as described in any one of implementation schemes 1-84 to a subject in need.

[0110] Implementation scheme 86 is a method of administering engineered cells, cell populations or pharmaceutical compositions as described in any one of implementation schemes 1-84 to a subject as adoptive cell transfer (ACT) therapy.

[0111] Implementation scheme 87 is a method of administering engineered cells, cell populations or pharmaceutical compositions as described in any one of implementation schemes 1-84 to a subject as an immunotherapy.

[0112] Embodiment 88 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 1-84, wherein the engineered cell, cell population, or pharmaceutical composition is used as an ACT therapy.

[0113] Implementation scheme 89 is a method for treating a disease or condition, the method comprising administering to a subject in need an engineered cell, cell population or pharmaceutical composition as described in any one of implementation schemes 1-84.

[0114] Embodiment 90 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 3-89, wherein the guide RNA is provided to the cell in a vector.

[0115] Embodiment 91 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 10-90, wherein the nucleic acid encoding the RNA-guided DNA binder is provided to the cell in the same vector as the guide RNA.

[0116] Embodiment 92 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 1-91, wherein an exogenous nucleic acid is provided to the cell, optionally in a carrier.

[0117] Implementation scheme 93 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of implementation schemes 90-92, wherein the vector is a viral vector.

[0118] Implementation scheme 94 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 93, wherein the carrier is AAV.

[0119] Embodiment 95 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-94, wherein the gene modification inhibits the expression of the gene in which the gene modification is present.

[0120] Embodiment 96 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of Embodiments 1-95, wherein the gene modification inhibits the expression of the TGFβR2 gene.

[0121] Embodiment 97 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-96, wherein the engineered cells have reduced surface expression of TGFβR2 protein compared to unmodified cells.

[0122] Implementation scheme 98 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 97, wherein the cell surface expression of TGFβR2 protein is below the detection level.

[0123] Embodiment 99 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-98, wherein the cell comprises an exogenous nucleic acid encoding a target receptor expressed on the surface of the engineered cell.

[0124] Implementation scheme 100 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 99, wherein the target receptor is a T cell receptor (TCR).

[0125] Embodiment 101 is an engineered cell, cell population, pharmaceutical composition, or cell method as described in Embodiment 100, wherein the target receptor is WT1 TCR.

[0126] Embodiment 102 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiment 99, wherein the target receptor is a chimeric antigen receptor (CAR).

[0127] Embodiment 103 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-102, wherein the engineered cell further comprises a gene modification of one or more of the CIITA, HLA-A, HLA-B, or TRAC genes.

[0128] Embodiment 104 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-103, wherein the engineered cells further have reduced surface expression of one or more of MHC class II proteins, HLA-A, HLA-B, or TRAC compared to unmodified cells.

[0129] Embodiment 105 is an engineered cell, cell population, pharmaceutical composition, or method as described in Embodiments 103 or 104, wherein the engineered cell comprises: i. gene modifications within genomic coordinates chr6:29942891-29942915 or chr6:29942609-29942633 of the HLA-A gene; ii. gene modifications within genomic coordinates chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229 of the HLA-B gene; iii. Gene modifications within the genomic coordinates chr14:22547524-22547544, chr14:22550574-22550598, or chr14:22550544-22550568 of the TRAC gene; iv. Gene modifications within the genomic coordinates chr16:10906643-10906667 or chr16:10907504-10907528 of the CIITA gene; or v. A combination of two or more of (i)-(iv).

[0130] Embodiment 106 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 103-105, wherein the engineered cell comprises at least one genetic modification, said genetic modification being: (i) within genomic coordinates targeted by an HLA-A guide RNA containing a guide sequence of SEQ ID NO: 403 or 404; (ii) within genomic coordinates targeted by an HLA-B guide RNA containing a guide sequence of SEQ ID NO: 406, 405, or 407; (iii) within genomic coordinates targeted by a TRAC guide RNA containing a guide sequence of SEQ ID NO: 413, 408, or 409; (iv) within genomic coordinates targeted by a CIITA guide RNA containing a guide sequence of SEQ ID NO: 402 or 401; or (v) a combination of two or more of (i)-(iv).

[0131] Implementation scheme 106.1 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of implementation schemes 103-106, wherein the engineered cell comprises gene modifications in the HLA-A gene, gene modifications in the HLA-B gene, gene modifications in the TRAC gene and gene modifications in the CIITA gene.

[0132] Implementation scheme 106.2 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of implementation schemes 103-106 and 106.1, wherein the engineered cell comprises: i. gene modification within genomic coordinates chr6:29942891-29942915 of the HLA-A gene; ii. gene modification within genomic coordinates chr6:31355222-31355246 of the HLA-B gene; iii. gene modification within genomic coordinates chr14:22547524-22547544 of the TRAC gene; and iv. gene modification within genomic coordinates chr16:10906643-10906667 of the CIITA gene.

[0133] Implementation scheme 106.3 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of implementation schemes 103-106, 106.1, and 106.2, wherein the engineered cell comprises: i. gene modification within genomic coordinates chr6:29942891-29942915 of the HLA-A gene; ii. gene modification within genomic coordinates chr6:31355222-31355246 of the HLA-B gene; iii. gene modification within genomic coordinates chr14:22547524-22547544 of the TRAC gene; iv. gene modification within genomic coordinates chr16:10906643-10906667 of the CIITA gene; and v. gene modification within genomic coordinates chr3:30674205-30674229 of the TGFβR2 gene.

[0134] Implementation scheme 106.4 is an engineered human cell, said engineered human cell comprising gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFβR2 gene, and gene modifications within the genomic coordinates chr14:22547524-22547544 of the TRAC gene.

[0135] Embodiment 107 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-106 and 106.1-106.4, wherein the engineered cell is an immune cell.

[0136] Implementation scheme 108 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 107, wherein the engineered cell is a monocyte, macrophage, mast cell, dendritic cell or granulocyte.

[0137] Implementation scheme 109 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 107, wherein the engineered cell is a lymphocyte.

[0138] Implementation scheme 110 is an engineered cell, cell population, pharmaceutical composition or method as described in implementation scheme 109, wherein the engineered cell is a T cell.

[0139] Embodiment 111 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-107 and 106.1-106.4, wherein the cell is a CD4+ T cell or a CD8+ T cell. Embodiment 112 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-107 and 106.1-106.4, wherein the cell is a memory T cell.

[0140] Embodiment 113 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-112 and 106.1-106.4, wherein the cell is a primary cell.

[0141] Embodiment 114 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-113 and 106.1-106.4, wherein the cell is a tissue-specific primary cell.

[0142] Embodiment 115 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-114 and 106.1-106.4, wherein the cell is an activated cell.

[0143] Embodiment 116 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-114 and 106.1-106.4, wherein the cell is an inactive cell.

[0144] Embodiment 117 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-116 and 106.1-106.4, wherein the cell is an allogeneic cell.

[0145] Embodiment 118 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of Embodiments 1-106 and 106.1-106.4, wherein the cell is a stem cell.

[0146] Implementation scheme 119 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of implementation schemes 1-118 and 106.1-106.4, said engineered cell, cell population, pharmaceutical composition or method being administered to a subject as an adoptive cell transfer (ACT) therapy.

[0147] Implementation scheme 120 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of implementation schemes 1-118 and 106.1-106.4, wherein the engineered cell, cell population, pharmaceutical composition or method is used to treat a subject with cancer.

[0148] Implementation scheme 121 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of implementation schemes 1-118 and 106.1-106.4, wherein the engineered cell, cell population, pharmaceutical composition or method is used to treat a subject suffering from an infectious disease.

[0149] Implementation scheme 122 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of implementation schemes 1-118 and 106.1-106.4, wherein the engineered cell, cell population, pharmaceutical composition, or method is used to treat a subject suffering from an autoimmune disease. Embodiment 123 is a population or pharmaceutical composition as described in any one of Embodiments 17-122, wherein, as measured by flow cytometry, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cell population is TGFβR2 negative.

[0150] Embodiment 124 is a population or pharmaceutical composition as described in any one of Embodiments 17-123, wherein, as measured by next-generation sequencing (NGS), at least 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cell population contains the genetic modification in the TGFβR2 gene.

[0151] A. Definition Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings: As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if the order is important in the particular context. Continuing this example, it explicitly includes combinations containing repetitions of one or more items or items, such as BB, AAA, AAB, BBC, CBBA, CABA, etc. Those skilled in the art will understand that, unless the context otherwise makes it obvious, there is generally no limit to the number of items or items in any combination.

[0152] As used herein, the term “kit” refers to a package of related components, such as one or more polynucleotides or compositions, and one or more related materials, such as delivery devices (e.g., syringes), solvents, solutions, buffers, instructions, or desiccants.

[0153] As used herein, an "allogeneic" cell refers to a cell derived from a donor subject of the same species as the recipient subject, wherein the donor and recipient subjects are genetically different, for example, having different genes at one or more loci. Thus, for example, the cell is allogeneic to the subject to which the cell is to be administered. As used herein, cells removed or separated from a donor and not reintroduced into the original donor are considered allogeneic cells.

[0154] As used herein, "autologous" cells refer to cells derived from the same subject into whom the material is later reintroduced. Therefore, for example, if cells are removed from a subject and then reintroduced into the same subject, the cells are considered autologous.

[0155] As used herein in the context of proteins, the term "TGFβR2" or "TGFBR2" refers to a transmembrane protein with a protein kinase domain that forms a heterodimeric complex with transforming growth factor β (TGF-β) receptor type 1 and binds to TGF-β. As used herein in the context of nucleic acids, the term "TGFβR2" or "TGFBR2" refers to the gene encoding the protein molecule of transforming growth factor β (TGF-β) receptor type 2. The Human Genome Project has accession number NC_000003.12 (30606356..30694142).

[0156] As used herein, the term "within genomic coordinates" includes the boundaries of a given range of genomic coordinates. For example, if chr6:29942854-chr6:29942913 is given, then the coordinates chr6:29942854-chr6:29942913 are covered. Throughout this application, the genomic coordinates referenced are based on genomic annotations from the human genome GRCh38 (also known as hg38) assembly from the Genome Reference Consortium, available on the website of the National Center for Biotechnology Information. Tools and methods for converting genomic coordinates between assemblies are known in the art and can be used to convert genomic coordinates provided herein to corresponding coordinates in another assembly of the human genome, including conversion to earlier assemblies generated by the same institution or using the same algorithm (e.g., from GRCh38 to GRCh37), and conversion to assemblies generated by different institutions or algorithms (e.g., from GRCh38 to NCBI33, generated by the International Human Genome Sequencing Consortium). Available methods and tools known in the art include, but are not limited to, the NCBIGENome Remapping Service, available at the National Center for Biotechnology Information; UCSC LiftOver, available at the UCSC Genome Brower; and Assembly Converter, available at Ensembl.org.

[0157] "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds containing nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers as analogs thereof. The nucleic acid "backbone" can consist of a variety of bonds, including one or more sugar-phosphodiester bonds, peptide-nucleic acid bonds ("peptide nucleic acid" or PNA; PCT No. WO 95 / 32305), thiophosphate bonds, methylphosphonate bonds, or combinations thereof. The sugar moiety of a nucleic acid can be ribose, deoxyribose, or a similar compound with substitutions, such as 2'-methoxy, 2'-halogen, or 2'-O-(2-methoxyethyl)(2'-O-moe) substitutions. RNA can contain one or more deoxyribonucleotides, for example as modifications, and similarly, DNA can contain one or more ribonucleotides. The nitrogenous base can be a conventional base (A, G, C, T, U), its analogues (e.g., modified uridines, such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine or others); inosine; or a derivative of a purine or pyrimidine (e.g., N...).4 -Methyldeoxyguanosine, denitropurine or azapurine, denitropyrimidine or azapyrimidine, pyrimidine bases with substituents at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with substituents at the 2, 6 or 8 position, 2-amino-6-methylaminopurine, O 6 -Methylguanine, 4-thiopyrimidine, 4-aminopyrimidine, 4-dimethylhydrazine-pyrimidine and O 4 -alkyl-pyrimidine; US Patent No. 5,378,825 and PCT No. WO 93 / 13121). For general discussion, see [link to general discussion]. The Biochemistry of the Nucleic Acids 5-36, Adams et al., eds., 11th ed., 1992. Nucleic acids may include one or more “base-free” residues, wherein the backbone does not include nitrogenous bases at one or more positions of the polymer (US Patent No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases, and linkages, or may include conventional components and substitutions (e.g., conventional nucleosides with a 2'-methoxy substituent, or polymers containing conventional nucleosides and one or more nucleoside analogs). Nucleic acids include “locked nucleic acids” (LNAs), which are analogs containing one or more LNA nucleotide monomers in which a bicyclic furanose unit is locked in RNA in a sugar-mimicking conformation, which enhances hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004). Biochemistry 43(42):13233-41). Nucleic acids include “non-locked nucleic acids”, which are capable of regulating thermodynamic stability and also provide nuclease stability. RNA and DNA have different sugar moieties and may differ due to the presence of uracil or its analogues in RNA and thymine or its analogues in DNA.

[0158] As used herein, "peptide" refers to a polymeric compound containing amino acid residues that can adopt a three-dimensional conformation. Peptides include, but are not limited to, enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid-binding proteins, and antibodies. Peptides may, but do not necessarily, contain post-translational modifications, non-natural amino acids, or prosthetic groups.

[0159] The terms “guide RNA,” “gRNA,” and simply “guide RNA” are used interchangeably in this document to refer to a single guide RNA, or a combination of crRNA and trRNA (also known as tracrRNA). crRNA and trRNA can associate as a single RNA strand (as a single guide RNA, sgRNA) or, for example, as two separate RNA strands (dgRNA). “Guide RNA” or “gRNA” refers to each type. trRNA can be a naturally occurring sequence or a modified or mutated trRNA sequence.

[0160] As used in this article, "guide sequence" refers to a sequence within the guide RNA that is complementary to the target sequence and has the function of guiding the guide RNA to the target sequence for binding or modification (e.g., cleavage) via an RNA-guided DNA binder.

[0161] In Neisseria meningitidis ( Neisseria meningitides In the case of Cas9 (i.e., NmeCas9 (NmeCas9)) and related Cas9 homologs / orthologs, the guide sequence can be 19, 20, 21, preferably 22, 23, or 24 nucleotides long, or 20-25 nucleotides long. In some embodiments, for example, the target sequence is located in a gene or on a chromosome and is complementary to the guide sequence. In some embodiments, the complementarity or identity between the guide sequence and its corresponding target sequence is at least 80%, 85%, preferably 90%, or 95%. In some embodiments, the guide sequence and the target region can be 100% complementary or identical. In other embodiments, the guide sequence and the target region can contain at least one mismatch, i.e., one different or non-complementary nucleotide, depending on the reference sequence. For example, the guide sequence and the target sequence can contain 1-2, preferably no more than 1 mismatch, wherein the total length of the target sequence is 19, 20, 21, 22, preferably 23, or 24 nucleotides, or more. In some embodiments, the guide sequence and target region may contain one to two mismatches, wherein the guide sequence contains at least 24 or more nucleotides. In some embodiments, the guide sequence and target region may contain one to two mismatches, wherein the guide sequence contains 24 nucleotides. That is, the guide sequence and target region may form a double-stranded region having at least 2X or more base pairs. In some embodiments, the double-stranded region may include one to two mismatches such that the guide strand and target sequence are not perfectly complementary. Mismatch locations are known in the art, and as provided, for example, distal mismatches in PAM tend to be more tolerant than proximal matches in PAM. Mismatch tolerance at other locations is known in the art (see, for example, Edraki et al., 2019. Mol. Cell, 73:1-13).

[0162] For example, the Nme guide sequence can be 19, 20, 21, preferably 22, 23, or 24 nucleotides long, such that in some embodiments, the Nme Cas9 guide sequence comprises at least 22, 23, or 24 consecutive nucleotides of the sequences provided in Table 2. In some embodiments, the guide sequence and the target sequence can be 100% complementary or identical. In other embodiments, the guide sequence and the target sequence can contain at least one mismatch, i.e., one different or non-complementary nucleotide, depending on the reference sequence. For example, the guide sequence and the target sequence can contain 1-2, preferably no more than 1, mismatch, wherein the total length of the target sequence is 19, 20, 21, 22, preferably 23, or 24 nucleotides, or more. In some embodiments, the guide sequence and the target region can contain 1 to 2 mismatches, wherein the guide sequence comprises at least 24 or more nucleotides. In some embodiments, the guide sequence and the target sequence can contain 1-2 mismatches, wherein the guide sequence comprises 24 nucleotides. In other words, the guide sequence and the target sequence can form a double-stranded region with 24 or more base pairs. In some embodiments, the double-stranded region may include one or two mismatches, such that the guide sequence and the target sequence are not perfectly complementary. Mismatch locations are known in the art; for example, distal mismatches in PAM tend to be more tolerant than proximal matches. Mismatch tolerance at other locations is known in the art (see, for example, Edraki et al., 2019. Mol. Cell, 73:1-13).

[0163] As defined by the guide sequence of the guide RNA, the target sequence of the RNA-guided DNA binder can be present on either the positive or negative strand. The tables and other disclosures provided herein may list genomic coordinates or locations within the nucleotide sequence that serve as the target sequence. It should be understood that, as defined by the genomic coordinates or locations within the nucleotide sequence, the guide can be complementary to either the positive or negative strand of the DNA. The sequence complementary to the guide depends on the presence of the appropriate PAM of the RNA-guided DNA-binding protein on the opposite strand. Thus, in some embodiments, when the guide sequence binds to the inverse complementary sequence of the target sequence, i.e., the guide sequence is identical to certain nucleotides of the sense (positive) strand of the target sequence (when the PAM is present in the sense strand), but the T in the guide sequence is replaced by U.

[0164] As used herein, "RNA-guided DNA binder" means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and dependent on the presence of PAM and the sequence of the guide RNA. Exemplary RNA-guided DNA binders include Cas lysins / nicking enzymes and their inactivated forms ("dCas DNA binders"). As used herein, "Cas nuclease" or "Cas9 protein" encompasses Cas lysins, Cas nicking enzymes, and dCas DNA binders. dCas DNA binders can be dead nucleases containing non-functional nuclease domains (RuvC or HNH domains). In some embodiments, Cas lysins or Cas nicking enzymes encompass dCas DNA binders modified to allow DNA cleavage (e.g., via fusion with a FokI domain). Cas lysins / nicking enzymes and dCas DNA binders include the Csm or Cmr complex of the type III CRISPR system, its Cas10, Csm1 or Cmr2 subunits, the cascade complex of the type I CRISPR system, its Cas3 subunit, and type II Cas nucleases.

[0165] As used herein, “class 2 Cas nucleases” are single-stranded polypeptides with RNA-guided DNA-binding activity. Class 2 Cas nucleases include class 2 Cas lyases / nicking enzymes (e.g., H840A or D10A variants of Spy Cas9, and D16A and H588A of Nme Cas9 (e.g., Nme2Cas9)) that further have RNA-guided DNA lyase or nicking enzyme activity; and class 2 dCas DNA binders in which the lyase / nicking enzyme activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and their modifications. The Cpf1 protein (Zetsche et al., Cell, 163: 1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated in its entirety by reference. See, for example, Zetsche, Tables S1 and S3. See, for example, Makarova et al., NatRev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0166] Several Cas9 orthologs have been obtained from Neisseria meningitidis (Esvelt et al., NAT. METHODS, Vol. 10, 2013, 1116-1121; Hou et al., PNAS, Vol. 110, 2013, pp. 15644-15649) (Nme1Cas9, Nme2Cas9, and Nme3Cas9). The Nme2Cas9 ortholog functions effectively in mammalian cells, recognizes N4CC PAM, and can be used for in vivo editing using homologous gRNAs (Ran et al., NATURE, Vol. 520, 2015, pp. 186-191; Kim et al., NAT. COMMUN., Vol. 8, 2017, p. 14500). Nme2Cas9 can be specific and selective, for example, capable of low-off-target editing (Lee et al., MOL. THER., Vol. 24, 2016, pp. 645–654; Kim et al., 2017). See also WO / 2020081568 (e.g., pp. 28 and 42), which describes the Nme2Cas9 D16A nickase, the contents of which are hereby incorporated in their entirety by reference. Throughout this text, “NmeCas9” or “NmeCas9” is generic and encompasses any type of NmeCas9, including Nme1Cas9, Nme2Cas9, and Nme3Cas9.

[0167] Exemplary nucleotide and polypeptide sequences of the Cas9 molecule are provided below. Methods for identifying alternative nucleotide sequences (including alternative naturally occurring variants) encoding the Cas9 polypeptide sequence are known in the art. Sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the Cas9 nucleic acid sequences or nucleic acid sequences encoding amino acid sequences provided herein are also covered. In some embodiments, the nucleotide sequence encoding the Cas9 amino acid sequence is not a naturally occurring Cas9 nucleotide sequence. Sequences having at least 95%, 96%, 97%, 98%, or 99% identity with any of the Cas9 amino acid sequences provided herein are also covered. In some embodiments, the Cas9 amino acid sequence is not a naturally occurring Cas9 sequence.

[0168] Table 10 provides exemplary open reading frames and amino acid sequences for Cas9 (SEQ ID NO: 802-810, 832-834) and uracil glycosidase inhibitors (SEQ ID NO: 823-826, 859, 860).

[0169] As used herein, the term "editor" refers to an agent containing a polypeptide capable of modifying a DNA sequence. In some embodiments, the editor is a lysin, such as Cas9 lysin. In some embodiments, the editor is capable of deaminating bases within a DNA molecule, and it may be referred to as a base editor. In some embodiments, the editor is capable of deaminating cytosine (C) in DNA. In some embodiments, the editor is a fusion protein containing an RNA-guided nicking enzyme fused to cytidine deaminase. In some embodiments, the editor is a fusion protein containing an RNA-guided nicking enzyme fused to APOBEC3A deaminase (A3A). In some embodiments, the editor contains a Cas9 nicking enzyme fused to APOBEC3A deaminase (A3A). In some embodiments, the editor is a fusion protein containing an RNA-guided nicking enzyme fused to both cytidine deaminase and UGI. In some embodiments, the editor lacks UGI. The exemplary editor used in this document can be described in WO2022125968, published on June 16, 2022, the contents of which are incorporated herein by reference. The exemplary editor may be a single polypeptide containing Homo sapiens ( ) linked via an XTEN linker to the Neisseria meningitidis-D16A Cas9 nickase. H. sapiens APOBEC3A. This article provides the mRNA encoding the above (e.g., SEQ ID NO: 801).

[0170] As used herein, “cytidine deaminase” refers to a polypeptide or polypeptide complex that possesses cytidine deaminase activity, catalyzing the hydrolytic deamination of cytidine or deoxycytidine, typically producing uridine or deoxyuridine. Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and particularly enzymes of the APOBEC family (enzymes of the APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups), activation-induced cytidine deaminases (AID or AICDA), and CMP deaminases (see, for example, Conticello et al., Mol. Biol. Evol. 22:367-77, 2005; Conticello, Genome Biol. 9:229, 2008; Muramatsu et al., J. Biol. Chem. 274: 18470-6, 1999; Carrington et al., Cells 9:1690(2020)).

[0171] As used herein, the term "APOBEC3" refers to the APOBEC3 protein, such as the APOBEC3 protein expressed by any of the seven genes (A3A-A3H) at the human APOBEC3 locus. APOBEC3 can have catalytic DNA or RNA editing activity. The amino acid sequence of APOBEC3A has been described (UniPROT Accession ID: p31941) and is included herein as SEQ ID NO: 850. In some embodiments, the APOBEC3 protein is the human APOBEC3 protein or the wild-type protein. Variants include proteins having sequences that differ from the wild-type APOBEC3 protein due to one or more mutations (i.e., substitution, deletion, insertion), such as one or more single-point substitutions. For example, a shortened APOBEC3 sequence may be used, for instance, by deleting several N-terminal or C-terminal amino acids, preferably deleting one to four amino acids from the C-terminus of the sequence. As used herein, the term "variant" refers to allelic variants, splicing variants, and natural or artificial mutants homologous to the APOBEC3 reference sequence. The variant is "functional" because it exhibits catalytic activity for DNA editing. In some embodiments, APOBEC3 (such as human APOBEC3A) has wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, APOBEC3 (such as human APOBEC3A) has asparagine at amino acid position 57 (as numbered in the wild-type sequence).

[0172] As used herein, a “nicking enzyme” is an enzyme that produces single-strand breaks (also called “nicks”) in double-stranded DNA (i.e., cuts one strand of the DNA double helix but not the other). As used herein, “RNA-guided DNA nicking enzyme” means a polypeptide or polypeptide complex having DNA nicking enzyme activity, wherein the DNA nicking enzyme activity is sequence-specific and depends on the RNA sequence. Exemplary RNA-guided DNA nicking enzymes include Cas nicking enzymes. Cas nicking enzymes include the Csm or Cmr complex of a type III CRISPR system, its Cas10, Csm1, or Cmr2 subunits, the cascade complex of a type I CRISPR system, its Cas3 subunit, and nicking enzyme forms of class 2 Cas nucleases. Class 2 Cas nicking enzymes include variants in which only one of the two catalytic domains is inactivated, said variants having RNA-guided DNA nicking enzyme activity. Class 2 Cas nickases include polypeptides in which the HNH or RuvC catalytic domain is inactivated, such as Cas9, for example Cas9 (e.g., H840A, D10A or N863A variants of SpyCas9 or D16A variant of NmeCas9). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domains or RuvC or RuvC-like domains of Neisseria meningitidis include Nme2Cas9 D16A (HNH nickase) and Nme2Cas9 H588A (RuvC nickase), Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., variants N497A, R661A, Q695A, Q926A), HypaCas9 (e.g., variants N692A, M694A, Q695A, H698A), eSPCas9(1.0) (e.g., variants K810A, K1003A, R1060A), and eSPCas9(1.1) (e.g., variants K848A, K1003A, R1060A) proteins and their modifications. The Cpf1 protein (Zetsche et al., Cell, 163: 1-13 (2015)) is homologous to Cas9 and contains a RuvC-like protein domain. Zetsche's Cpf1 sequence is incorporated in its entirety by reference. See, for example, Zetsche, Tables S1 and S3. "Cas9" encompasses *S. pyogenes* (Spy) Cas9, the Cas9 variants listed herein, and their equivalents. See, for example, Makarova et al., *Nat Rev Microbiol*, 13(11): 722-36 (2015); Shmakov et al., *Molecular Cell*, 60:385-397 (2015).

[0173] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising polypeptides derived from at least two different proteins or sources. A polypeptide may be located at the N-terminal (N-terminal) portion or the C-terminal (C-terminal) portion of the fusion protein, thereby forming an "N-terminal fusion protein" or a "C-terminal fusion protein," respectively. Any protein presented herein can be produced by any method known in the art. For example, the proteins presented herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0174] As used herein, the term "linker" refers to a chemical group or molecule that connects two adjacent molecules or portions. Typically, a linker is located between or on either side of two groups, molecules, or other portions and is covalently linked to each other. In some embodiments, the linker is one or more amino acids (e.g., peptides or proteins), such as the 16-amino acid residue "XTEN" linker or a variant thereof (see, for example, examples; and Schellenberger et al., A recombinant polypeptide extends the in vivo half-life of peptides and proteins in atunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequences SGSETPGTSESATPES (SEQ ID NO: 901), SGSETPGTSESA (SEQ ID NO: 902), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 903).

[0175] As used herein, the terms “uracil glycosidase inhibitor,” “uracil-DNA glycosidase inhibitor,” or “UGI” refer to proteins that inhibit the uracil-DNA glycosidase (UDG) base excision repair enzyme (e.g., UniPROT ID: P14739; SEQ ID NO: 859).

[0176] As used herein, an "open reading frame" or "ORF" of a gene refers to a sequence of codons that specify the amino acid sequence of the protein encoded by the gene. An ORF begins with a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon (e.g., TAA, TAG, or TGA in DNA or UAA, UAG, or UGA in RNA).

[0177] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA along with an RNA-guided DNA binder, such as a Cas nuclease, for example a Cas lyase, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA directs the RNA-guided DNA binder (such as Cas9) to a target sequence, and the guide RNA hybridizes to the target sequence and the binder binds to the target sequence; where the binder is a lyase or a nickase, cleavage or nicking can occur after binding.

[0178] As used herein, a first sequence is considered to "contain a sequence with at least X% identity to the second sequence" if an alignment of the first and second sequences shows that X% or more positions in the entire second sequence match the first sequence. For example, the sequence AAGA contains a sequence with 100% identity to the sequence AAG because the alignment would result in 100% identity due to matching all three positions in the second sequence. Differences between RNA and DNA (generally uridine exchanged for thymidine and vice versa) and the presence of nucleoside analogs (such as modified uridine) do not result in differences in identity or complementarity between polynucleotides, provided that the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., for all of thymidine, uridine, or modified uridine, it is adenosine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as complement). Therefore, for example, the sequence 5'-AXG (where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine) is considered to have 100% identity with AUG because both are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand which algorithm and parameter settings are suitable for a given sequence pair to be aligned; for sequences generally of similar length and expected identity (>50% for amino acids or >75% for nucleotides), the Needleman-Wunsch algorithm with its preset settings provided by EBI on the www.ebi.ac.uk web server is generally appropriate.

[0179] In this document, “mRNA” refers to a polynucleotide that contains an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation into ribosomes and aminoacylated tRNA). mRNA may contain a phosphate-sugar backbone comprising ribose residues or analogues thereof, such as 2'-methoxyribose residues. In some embodiments, the sugars in the mRNA phosphate-sugar backbone are substantially composed of ribose residues, 2'-methoxyribose residues, or combinations thereof.

[0180] As used herein, “insertion / deletion” refers to an insertion or deletion mutation consisting of multiple nucleotides that are inserted, deleted, or inserted and deleted in a target nucleic acid, such as at a double-strand break (DSB) site. As used herein, when an insertion / deletion results in an insertion, the insertion is a random insertion at a DSB site and is generally not guided by or based on a template sequence.

[0181] As used herein, “reduced or eliminated” expression of a protein on a cell refers to a partial or complete loss of protein expression relative to unmodified cells. In some embodiments, the surface expression of a protein on a cell is measured by flow cytometry, and “reduced” or “eliminated” surface expression relative to unmodified cells is demonstrated by a reduction in fluorescence signal after staining with the same antibody targeting the protein. Cells that show “reduced” or “eliminated” surface expression of a protein relative to unmodified cells by flow cytometry can be described as having “negative” expression for that protein, as demonstrated by a fluorescence signal similar to that of cells stained with an isotype control antibody. The “reduction” or “elimination” of protein expression can be measured using other techniques known in the art, utilizing appropriate controls known to those skilled in the art.

[0182] As used herein, “knockdown” refers to, for example, a reduction in the expression of a specific gene product (e.g., protein, mRNA, or both) compared to the expression of an unedited target sequence. Protein knockdown can be measured by detecting the total cellular amount of protein from a sample (such as a tissue, fluid, or cell population of interest). Protein knockdown can also be measured by measuring protein substitutes, markers, or activities. Methods for measuring mRNA knockdown are known and involve analyzing mRNA isolated from the sample of interest. In some embodiments, “knockdown” can refer to some loss of expression of a specific gene product, such as a reduction in the amount of transcribed mRNA or a reduction in the amount of protein expressed by cells or cell populations (including in vivo populations, such as those found in tissues).

[0183] As used in this article, "knockout" or "KO" refers to the loss of expression of a specific gene or protein in a cell. Knockout can cause expression to drop below the detectable level. Knockout can be measured by detecting the total amount of protein in a cell, tissue, or cell population.

[0184] As used herein, "target sequence" or "genomic target sequence" refers to a nucleic acid sequence in a target gene that is complementary to the guide sequence of the gRNA. The interaction between the target sequence and the guide sequence directs the binding of an RNA-guided DNA binder within the target sequence and potentially causes cleavage or splitting (depending on the activity of the binder).

[0185] As used herein, the term “subject” is intended to include living organisms that can elicit an immune response, including, for example, mammals, primates, and humans.

[0186] As used herein, “treatment” means any administration or application of a therapeutic agent to a subject’s disease or condition, and includes suppressing the disease, halting the progression of the disease, alleviating one or more symptoms of the disease, curing the disease, or preventing one or more symptoms of the disease (including recurrence of symptoms).

[0187] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention has been described in conjunction with the illustrated embodiments, it will be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the invention as defined in the appended claims and the included embodiments.

[0188] Before describing this doctrine in detail, it should be understood that this disclosure is not limited to specific compositions or process steps, as they can vary. It should be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “described” as used in this specification and the appended claims include plural references. Thus, for example, reference to “a conjugate” includes multiple conjugates, and reference to “a cell” includes multiple cells (e.g., a cell population), etc.

[0189] Numerical ranges include the numerical values ​​that define the range. Taking into account significant figures and measurement-related errors, measured and measurable values ​​should be understood as approximate values.

[0190] Furthermore, the use of "comprise / comprises / comprising," "contain / contains / containing," and "include / includes / including" is not intended to be limiting. It should be understood that the foregoing general and detailed descriptions are merely exemplary and illustrative, and not doctrinal limitations. Unless specifically indicated in the specification, embodiments described as "comprise" various components are also contemplated as consisting of "by" or "substantially by" the components described; embodiments described as consisting of various components are also contemplated as "containing" or "substantially by" the components described; and embodiments described as "substantially by" various components are also contemplated as "by" or "containing" the components described (this interchangeability does not apply to the use of these terms in the claims).

[0191] Unless the context clearly indicates otherwise, the term "or" is used in the inclusive sense, that is, equivalent to "and / or".

[0192] When used before a list, the term "about" modifies each member of the list. The term "about" should be understood to cover variations or errors permissible in the art, such as a deviation of 2 standard deviations from the average or sensitivity of the method used to make the measurement. When "about" appears before the first value in a series, it can be understood to modify each value in said series.

[0193] The range should be understood to include the numerical value at the end of the range and all logical values ​​in between. For example, 5-10 nucleotides should be understood as 5, 6, 7, 8, 9, or 10 nucleotides, while 5-10% should be understood as containing 5% and all possible values ​​up to 10%.

[0194] It will be clearly understood that, even without a specific upper limit, at least 17 nucleotides in a 20-nucleotide sequence should be understood to include 17, 18, 19, or 20 nucleotides of the provided sequence, thus providing an upper limit. Similarly, even without a specific lower limit, at most 3 nucleotides will be understood to cover 0, 1, 2, or 3 nucleotides, thus providing a lower limit. When “at least,” “at most,” or other similar language modifies a numerical value, it can be understood to modify each value in the series.

[0195] As used herein, “not greater than” or “less than” should be understood as a value adjacent to a phrase and a logically low value or integer, logically to zero in context. For example, the double-stranded region of “not greater than 2 nucleotide base pairs” has 2, 1, or 0 nucleotide base pairs. When “not greater than” or “less than” appears before a series of values ​​or ranges, it should be understood as modifying each value in the series or range.

[0196] As used in this article, the range includes both the upper and lower limits.

[0197] If there is a conflict between the sequence in this application and the specified accession number or position in the accession number, the sequence in this application shall prevail.

[0198] As used herein, it should be understood that when the maximum value is expressed as 100% (e.g., 100% inhibition or 100% encapsulation), the value is limited by the detection method. For example, 100% inhibition should be understood as inhibition to a level below the detection level to be determined, and 100% encapsulation should be understood as the encapsulated material not being detectable outside the vesicle.

[0199] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. In the event of any material incorporated by reference that contradicts any terminology defined in this specification or any other express content herein, this specification shall prevail. While this doctrine has been described in conjunction with various embodiments, it is not intended to limit this doctrine to such embodiments. Rather, as those skilled in the art will understand, this doctrine encompasses various alternatives, modifications, and equivalents.

[0200] B. Genetically modified cells 1. Engineered cell composition This disclosure provides engineered cell compositions having reduced or eliminated surface expression of TGFβR2 protein relative to unmodified cells, and containing gene modifications in the TGFβR2 gene.

[0201] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of TGFβR2 protein relative to unmodified cells, and comprising a gene modification in the TGFβR2 gene, wherein the gene modification is located within genomic coordinates chr3:30606891-30691605. In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of TGFβR2 protein relative to unmodified cells, and comprising a gene modification in the TGFβR2 gene, wherein the gene modification comprises at least one nucleotide within genomic coordinates chr3:30606891-30691605. In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of TGFβR2 protein relative to unmodified cells, and comprising a gene modification in the TGFBR2 gene, wherein the gene modification is located within genomic coordinates chr3:30606891-30691605.

[0202] In some implementations, for each given range of genomic coordinates, the range may cover + / - 10 nucleotides at both ends of the specified coordinates. For example, if chr3:30606891-30606914 is given, then in some implementations, the genomic target sequence or gene modification may be located within chr3:30606891-30606914. In some implementations, for each given range of genomic coordinates, the range may cover + / - 5 nucleotides at either end of the range.

[0203] In some implementations, a given range of genomic coordinates may include target sequences on both strands of DNA (i.e., the positive (+) strand and the negative (+) strand).

[0204] Genetic modifications in the TGFβR2 gene are further described herein. In some embodiments, genetic modifications in the TGFβR2 gene include any one or more of the insertion, deletion, substitution, or deamination of at least one nucleotide in the target sequence.

[0205] The engineered cells described in this article can contain gene modifications in any TGFβR2 allele of the TGFβR2 gene. The TGFβR2 gene is located on chromosome 3.

[0206] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of the TGFβR2 protein relative to an unmodified cell, and containing genetic modifications in the TGFβR2 gene, wherein the genetic modifications include insertions / deletions, C-to-T substitutions, or A-to-G substitutions within any of the genomic coordinates listed in Table 2.

[0207] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of the TGFβR2 protein relative to an unmodified cell, and containing genetic modifications in the TGFβR2 gene, wherein the genetic modifications include insertions / deletions, C-to-T substitutions, or A-to-G substitutions within any of the genomic coordinates listed in Table 2, wherein the genetic modifications contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates.

[0208] In some embodiments, the gene modification comprises at least 5 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 1 consecutive nucleotide within the genomic coordinate system. In some embodiments, the gene modification comprises at least 2 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 3 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 4 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 5 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 6 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 7 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 8 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 9 consecutive nucleotides within the genomic coordinate system. In some embodiments, the gene modification comprises at least 10 consecutive nucleotides within the genomic coordinate system.

[0209] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of the TGFβR2 protein relative to an unmodified cell, and containing genetic modifications in the TGFβR2 gene, wherein the genetic modifications include insertions / deletions, C-to-T substitutions, or A-to-G substitutions within any of the genomic coordinates listed in Table 2, wherein the genetic modifications include at least one C-to-T substitution or at least one A-to-G substitution within the genomic coordinates.

[0210] In some embodiments, an engineered cell is provided in which TGFβR2 expression is reduced or eliminated by a gene editing system that binds to a TGFβR2 genomic target sequence comprising at least 5 consecutive nucleotides within any of the genomic coordinates listed in Table 2. In some embodiments, the TGFβR2 genomic target sequence comprises at least 10 consecutive nucleotides within the genomic coordinates. In some embodiments, the TGFβR2 genomic target sequence comprises at least 15 consecutive nucleotides within the genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binder, such as Neisseria meningitidis Cas9, or a base editor comprising a Neisseria meningitidis Cas9 nickase.

[0211] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFβR2 protein relative to unmodified cells, and includes a genetic modification of the TGFβR2 gene, wherein the genetic modification is located within the following genomic coordinates: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; chr3:30672133-30672157; chr3:30606891-30606915; chr3:3060689 2-30606916;chr3:30606896-30606920;chr3:30606897-30606921;chr3: 30606898-30606922;chr3:30606899-30606923;chr3:30606908-3060693 2;chr3:30606909-30606933;chr3:30606910-30606934;chr3:30606917- 30606941;chr3:30606958-30606982;chr3:30606959-30606983;chr3:306 06960-30606984;chr3:30606964-30606988;chr3:30606965-30606989;c hr3:30644900-30644924; chr3:30671667-30671691; chr3:30671670-306 71694;chr3:30671753-30671777;chr3:30671762-30671786;chr3:30671 766-30671790;chr3:30672034-30672058;chr3:30672126-30672150;chr3 :30672128-30672152;chr3:30672131-30672155;chr3:30672135-306721 59;chr3:30672139-30672163;chr3:30672140-30672164;chr3:30672140 -30672164;chr3:30672141-30672165;chr3:30672190-30672214;chr3:3 0672204-30672228;chr3:30672432-30672456;chr3:30672433-30672457;chr3:30672434-30672458; chr3:30674211-30674235; chr3:30688450-30688474; chr3:30688459-30688483; chr3:30688460-30688484; chr3:30688476-30688500; chr3:30688478-30688502; chr3:30688479-30688503; chr3:30691436-30691460; and chr3:30691581-30691605.

[0212] In some embodiments, an engineered cell is provided in which TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least five consecutive nucleotides selected from the following genomic coordinates: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; chr3:30672133-30672157; chr3:30606891-30606915; chr3:30606892-30606 916;chr3:30606896-30606920;chr3:30606897-30606921;chr3:306068 98-30606922;chr3:30606899-30606923;chr3:30606908-30606932;chr3 :30606909-30606933; chr3:30606910-30606934; chr3:30606917-306069 41;chr3:30606958-30606982;chr3:30606959-30606983;chr3:30606960 -30606984;chr3:30606964-30606988;chr3:30606965-30606989;chr3: 30644900-30644924;chr3:30671667-30671691;chr3:30671670-3067169 4;chr3:30671753-30671777;chr3:30671762-30671786;chr3:30671766 -30671790;chr3:30672034-30672058;chr3:30672126-30672150;chr3:3 0672128-30672152; chr3:30672131-30672155; chr3:30672135-3067215 9;chr3:30672139-30672163;chr3:30672140-30672164;chr3:30672140- 30672164;chr3:30672141-30672165;chr3:30672190-30672214;chr3:30 672204-30672228;chr3:30672432-30672456;chr3:30672433-30672457;chr3:30672434-30672458; chr3:30674211-30674235; chr3:30688450-30688474; chr3:30688459-30688483; chr3:30688460-30688484; chr3:30688476-30688500; chr3:30688478-30688502; chr3:30688479-30688503; chr3:30691436-30691460; and chr3:30691581-30691605.

[0213] In some embodiments, the TGFBR2 genomic target sequence comprises at least 10 consecutive nucleotides within genomic coordinates. In some embodiments, the TGFBR2 genomic target sequence comprises at least 15 consecutive nucleotides within genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binder, such as the Neisseria meningitidis Cas9 nickase.

[0214] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of the TGFBR2 protein relative to an unmodified cell, and containing a genetic modification in the TGFBR2 gene, wherein the genetic modification is located within the following genomic coordinates: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; and chr3:30672133-30672157. In some embodiments, an engineered cell is provided in which TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least five consecutive nucleotides selected from the following genomic coordinates: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; and chr3:30672133-30672157.

[0215] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of the TGFBR2 protein relative to an unmodified cell, and containing a genetic modification in the TGFBR2 gene, wherein the genetic modification is located within the following genomic coordinates: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; and chr3:30672133-30672157. In some embodiments, an engineered cell is provided in which TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least five consecutive nucleotides selected from the following genomic coordinates: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; and chr3:30672133-30672157.

[0216] In some embodiments, the TGFBR2 genomic target sequence comprises at least 10 consecutive nucleotides within genomic coordinates. In some embodiments, the TGFBR2 genomic target sequence comprises at least 15 consecutive nucleotides within genomic coordinates.

[0217] In some implementations, the TGFBR2 genomic target sequence contains at least 20, 21, 22, 23, or 24 consecutive nucleotides within genomic coordinates.

[0218] In some embodiments, the gene editing system includes a transcription activator-like effector nuclease (TALEN). In some embodiments, the gene editing system includes a zinc finger nuclease. In some embodiments, the gene editing system includes a CRISPR / Cas system, such as a class 2 system. In some embodiments, the gene editing system includes an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0219] Exemplary RNA-guided DNA binders are shown in Table 1 below.

[0220] Table 1. Exemplary RNA-guided DNA binders.

[0221] *Exemplary base editors based on deaminase-SpyCas9 nicking enzyme or deaminase-NmeCas9 nicking enzyme. It is evident that the specificity of the base editor (including PAM) will vary depending on its nicking enzyme.

[0222] In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder comprises the Cas9 protein. In some embodiments, the RNA-guided DNA binder is selected from one of the following: Streptococcus pyogenes Cas9, Neisseria meningitidis Cas9 (e.g., Nme2Cas9), Streptococcus thermophilus Cas9, Staphylococcus aureus Cas9, Neoculcium fragilis Cpf1, Aminococcus spp. Cpf1, Trichophyton spp. Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, Mad7 nuclease, ARCUS nuclease, and CasX. In some implementations, the RNA-guided DNA binder comprises a polypeptide selected from the following: Streptococcus pyogenes Cas9, Neisseria meningitidis Cas9 (e.g., Nme2Cas9), Streptococcus thermophilus Cas9, Staphylococcus aureus Cas9, Francisella neonicotinoides Cpf1, Aminococcus spp. Cpf1, Trichophyton spp. Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, and CasX.

[0223] In some implementations, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is Neisseria meningitidis Cas9, such as Nme2Cas9.

[0224] In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is *Streptococcus thermophilus* Cas9. In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is *Staphylococcus aureus* Cas9. In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is derived from *Francisella catarrhalis* (a novel culprit). F. novicida The RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is Cpf1 from the genus *C.* In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is Cpf1 from *C.* ND2006 of *C.* In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is Cas12a. In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is CasX.

[0225] In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is a C-to-T base editor. In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is an A-to-G base editor. In some embodiments, the base editor comprises a deaminase and an RNA-guided nicking enzyme. In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder comprises APOBEC3A deaminase (A3A) and an RNA-guided nicking enzyme. In some embodiments, the RNA-guided nicking enzyme comprises an NmeCas9 nicking enzyme.

[0226] In any of the above embodiments, the gene editing system comprises an RNA-guided DNA binder or a nucleic acid encoding the RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder comprises Cas9. In some embodiments, the RNA-guided DNA binder is Neisseria meningitidis or Nme2 Cas9. In some embodiments, the RNA-guided DNA binder is a base editor. In some embodiments, the base editor comprises a C-to-T deaminase and an RNA-guided nicking enzyme, such as Neisseria meningitidis or Nme2 Cas9 nicking enzyme. In some embodiments, the base editor comprises an A-to-G deaminase and an RNA-guided nicking enzyme, such as Neisseria meningitidis or Nme2 Cas9 nicking enzyme.

[0227] In some embodiments, the gene editing system further comprises a uracil glycosidase inhibitor (UGI), and the UGI and base editor are contained in a single polypeptide. In some embodiments, the gene editing system comprises a UGI, and the UGI and base editor are contained in different polypeptides. In some embodiments, the base editor comprises cytidine deaminase and an RNA-guided nickase. In some embodiments, cytidine deaminase, RNA-guided nickase, and UGI are contained in a single polypeptide. In some embodiments, cytidine deaminase, RNA-guided nickase, and UGI are contained in different polypeptides. In some embodiments, cytidine deaminase and RNA-guided nickase are contained in a single polypeptide, and wherein the UGI is contained in different polypeptides.

[0228] Engineered cells can be any of the exemplary cell types disclosed herein.

[0229] In some embodiments, this disclosure provides a pharmaceutical composition comprising any of the engineered cells disclosed herein. In some embodiments, the pharmaceutical composition comprises a population of any of the engineered cells disclosed herein. In some embodiments, as measured by flow cytometry, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cell population is pSAMD2 / 3 negative. In some embodiments, as measured by next-generation sequencing (NGS), at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cell population contains gene modifications in the TGFBR2 gene.

[0230] C. Methods and compositions for reducing or eliminating TGFBR2 surface expression This disclosure provides methods and compositions for reducing or eliminating the surface expression of the TGFBR2 protein relative to unmodified cells by genetically modifying the TGFBR2 gene. The resulting genetically modified cells may also be referred to herein as engineered cells. In some embodiments, the genetically modified (or engineered) cells may be starter cells for further genetic modification using the methods or compositions provided herein. In some embodiments, the cells are allogeneic cells. In some embodiments, cells with reduced or eliminated surface expression of the TGFBR2 protein can be used for immunotherapy. In some embodiments, cells with reduced or eliminated surface expression of the TGFBR2 protein can be used for adoptive cell transfer therapy. In some embodiments, editing of the TGFBR2 gene is combined with additional genetic modifications to produce cells suitable for allogeneic transplantation purposes.

[0231] In some embodiments, the method includes reducing the surface expression of TGFBR2 protein in cells relative to unmodified cells, comprising contacting the cells with a composition comprising: (a) a guide RNA comprising (i) a guide sequence selected from SEQ ID NO: 1-49; or (ii) at least 19, 20, 21, 22, 23, preferably 24 or 25 consecutive nucleotides selected from SEQ ID NO: 1-49; or (iii) a sequence selected from SEQ ID NO: The sequence 1-49 has at least 95%, 90% or 85% identity with the guide sequence; or (iv) a guide sequence that binds to a target site containing a genomic region listed in Table 2; or (v) a guide sequence that is complementary to at least 19, 20, 21, 22, 23, preferably 24 or 25 consecutive nucleotides of a genomic region listed in Table 2; or (vi) a guide sequence that has at least 95%, 90% or 85% identity with a sequence selected from (v); and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0232] In some embodiments, the method further includes contacting the cell with an RNA-guided DNA binder or a nucleic acid encoding the RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder comprises the Cas9 protein.

[0233] In some embodiments, the RNA-guided DNA binder is Neisseria meningitidis Cas9, such as Nme2Cas9. In some embodiments, the guide RNA is Nme Cas9 guide RNA.

[0234] In some implementations, the RNA-guided DNA binder contains a deaminase domain.

[0235] In some embodiments, the RNA-guided DNA binder is a C-to-T base editor. In some embodiments, the RNA-guided DNA binder is an A-to-G base editor. In some embodiments, the base editor comprises a deaminase and an RNA-guided nicking enzyme. In some embodiments, the RNA-guided DNA binder comprises APOBEC3A deaminase (A3A) and an RNA-guided nicking enzyme. In some embodiments, the RNA-guided nicking enzyme comprises NmeCas9 nicking enzyme.

[0236] In some implementations, the surface expression of the TGFBR2 protein (i.e., engineered cells) is thereby reduced or eliminated.

[0237] In some embodiments, the method includes manufacturing engineered human cells having reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, comprising contacting the cells with a composition comprising: (a) a guide RNA, the guide RNA comprising (i) a guide sequence selected from SEQ ID NO: 1-49; or (ii) at least 19, 20, 21, 22, 23, preferably 24 or 25 consecutive nucleotides selected from sequences selected from SEQ ID NO: 1-49; or (iii) a sequence containing (i) a guide sequence selected from SEQ ID NO: 1-49. The sequence 1-49 has at least 95%, 90%, or 85% identity with the guide sequence; or (iv) a guide sequence binding to a target site comprising a genomic region listed in Table 2; or (v) a guide sequence complementary to at least 19, 20, 21, 22, 23, preferably 24, or 25 consecutive nucleotides of a genomic region listed in Table 2; or (vi) a guide sequence having at least 95%, 90%, or 85% identity with a sequence selected from (v); and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the method further includes contacting the cell with the RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is Cas9. In some embodiments, the RNA-guided DNA binder is NmeCas9. In some embodiments, the guide RNA is... Nme Guide RNA. In some embodiments, the RNA-guided DNA binder comprises a deaminase domain. In some embodiments, the RNA-guided DNA binder comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase.

[0238] In some embodiments, the composition further comprises a uracil glycosidase inhibitor (UGI). In some embodiments, the composition comprises an RNA-guided DNA binder that converts cytosine (C) of a TGFBR2 genomic target sequence to thymine (T). In some embodiments, the composition comprises an RNA-guided DNA binder that converts adenosine (A) of a TGFBR2 genomic target sequence to guanine (G).

[0239] In some implementations, the surface expression of the TGFBR2 protein (i.e., engineered cells) is thereby reduced or eliminated.

[0240] In some embodiments, an engineered cell is provided, said engineered cell being produced by the methods described herein. In some embodiments, the compositions disclosed herein further comprise a pharmaceutically acceptable carrier. In some embodiments, a cell is provided, said cell being produced by a composition disclosed herein comprising a pharmaceutically acceptable carrier. In some embodiments, a composition comprising the cells disclosed herein is provided.

[0241] 1. TGFBR2 guide RNA The methods and compositions provided herein disclose guide RNAs that can be used to reduce or eliminate surface expression of the TGFBR2 protein. In some embodiments, such guide RNAs direct an RNA-guided DNA binder to a TGFBR2 genomic target sequence and may be referred to herein as "TGFBR2 guide RNAs". In some embodiments, the TGFBR2 guide RNA directs an RNA-guided DNA binder to a human TGFBR2 genomic target sequence. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence selected from SEQ ID NO: 1-49. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence selected from SEQ ID NO: 1-49.

[0242] In some embodiments, a composition is provided comprising the guide RNA and RNA-guided DNA binder described herein, or a nucleic acid encoding an RNA-guided DNA binder.

[0243] In some embodiments, a composition is provided comprising a single-guide RNA (sgRNA) comprising a guide sequence selected from SEQ ID NO: 1-49. In some embodiments, a composition is provided comprising the TGFBR2 sgRNA described herein and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0244] In some embodiments, a composition is provided comprising a TGFBR2 dual guide RNA (dgRNA) comprising a guide sequence selected from SEQ ID NO: 1-49. In some embodiments, a composition is provided comprising the TGFBR2 dgRNA described herein and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0245] In some embodiments, the TGFBR2 gRNA includes a guide sequence selected from any of SEQ ID NO: 1-49. Exemplary TGFBR2 target coordinate guide sequences are shown in Table 2 below (SEQ ID NO: 1-49). The guide sequences disclosed in this table may be unmodified, modified with the exemplary modification patterns shown in the table, or modified with different modification patterns disclosed herein or available in the art.

[0246] Table 2. Genomic coordinates and guide sequences of exemplary TGFBR2 guide RNAs

[0247] Table 3. Exemplary complete and modified guide RNAs

[0248] In some embodiments, the TGFBR2 guide RNA comprises the sequence of any of the guide RNA sequences shown in Tables 2-3. In some embodiments, the TGFBR2 gRNA comprises a guide sequence selected from any of SEQ ID NO: 1-49. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence of any of SEQ ID NO: 1-5. In some embodiments, the TGFBR2 guide RNA comprises the guide sequence of SEQ ID NO: 5.

[0249] In some embodiments, the TGFBR2 gRNA comprises a guide sequence selected from any of SEQ ID NO: 1-49. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence of at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NO: 1-49. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence having at least 95%, 90%, or 85% identity with a sequence selected from SEQ ID NO: 1-49. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence having at least 95%, 90%, or 85% identity with a sequence selected from SEQ ID NO: 1-49.

[0250] In some embodiments, the TGFBR2 guide RNA comprises a guide sequence comprising at least 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2. As used herein, at least 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates means, for example, at least 10 consecutive nucleotides within a genomic coordinate, wherein the genomic coordinate includes 10 nucleotides in the 5' direction and 10 nucleotides in the 3' direction within the range listed in Table 2. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence of at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence comprising 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence having at least 95%, 90%, or 85% identity with a sequence selected from sequences comprising 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence comprising 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2. In some implementations, the TGFBR2 guide RNA contains a guide sequence comprising at least 20 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2.

[0251] In some embodiments, a composition is provided comprising a base editor as described herein, the base editor comprising a cytidine deaminase and an RNA-guided nickase; and a TGFBR2 gRNA comprising: (i) a guide sequence selected from any of SEQ ID NO: 1-49; (ii) a guide sequence of at least 17, 18, 19, or 20 consecutive nucleotides of any of SEQ ID NO: 1-49; or (iii) a guide sequence having at least 95%, 90%, or 85% identity with any of SEQ ID NO: 1-49. In some embodiments, the RNA-guided nickase is an Nme2Cas9 nickase.

[0252] This article provides additional implementations of the TGFBR2 guide RNA, including, for example, exemplary modifications to the guide RNA.

[0253] 2. Gene modification of TGFBR2 In some embodiments, the methods and compositions disclosed herein genetically modify at least one nucleotide in the TGFBR2 gene in cells. Genetic modification encompasses modified populations generated through contact with a gene editing system (e.g., edited populations generated by Cas9 and TGFBR2 guide RNA, or edited populations generated by BC22 and TGFBR2 guide RNA).

[0254] In some embodiments, the gene modification is located within the genomic coordinates chr3:30606891-30691605. In some embodiments, the gene modification comprises at least one nucleotide within the genomic coordinates chr3:30606891-30691605.

[0255] In some embodiments, the gene modification is within any of the genomic coordinates listed in Table 2. In some embodiments, the gene modification comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides within any of the genomic coordinates listed in Table 2.

[0256] In some embodiments, the gene modification comprises at least one nucleotide selected from the following genomic coordinates: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; chr3:30672133-30672157; chr3:30606891-30606915; chr3:30606892-30606916; chr3:30606896-30606920; chr3:30606897-30606921; chr3:306 06898-30606922;chr3:30606899-30606923;chr3:30606908-30606932;c hr3:30606909-30606933; chr3:30606910-30606934; chr3:30606917-3060 6941;chr3:30606958-30606982;chr3:30606959-30606983;chr3:306069 60-30606984;chr3:30606964-30606988;chr3:30606965-30606989;chr3: 30644900-30644924;chr3:30671667-30671691;chr3:30671670-3067169 4;chr3:30671753-30671777;chr3:30671762-30671786;chr3:30671766-3 0671790; chr3:30672034-30672058; chr3:30672126-30672150; chr3:306 72128-30672152;chr3:30672131-30672155;chr3:30672135-30672159;ch r3:30672139-30672163; chr3:30672140-30672164; chr3:30672140-3067 2164;chr3:30672141-30672165;chr3:30672190-30672214;chr3:3067220 4-30672228;chr3:30672432-30672456;chr3:30672433-30672457;chr3:3 0672434-30672458;chr3:30674211-30674235;chr3:30688450-30688474;chr3:30688459-30688483; chr3:30688460-30688484; chr3:30688476-30688500; chr3:30688478-30688502; chr3:30688479-30688503; chr3:30691436-30691460; and chr3:30691581-30691605.

[0257] In some embodiments, the gene modification comprises at least one nucleotide selected from the following genomic coordinates: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; and chr3:30672133-30672157.

[0258] In some embodiments, modification of TGFBR2 comprises any one or more of the insertion, deletion, substitution, or deamination of at least one nucleotide in the target sequence. In some embodiments, modification of TGFBR2 comprises the insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, modification of TGFBR2 comprises the deletion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In other embodiments, modification of TGFBR2 comprises the insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In other embodiments, modification of TGFBR2 comprises the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, modification of TGFBR2 comprises insertion / deletion, which is generally defined in the art as an insertion or deletion of less than 1000 base pairs (bp). In some embodiments, modification of TGFBR2 involves insertion / deletion, resulting in a frameshift mutation in the target sequence. In some embodiments, modification of TGFBR2 involves substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, modification of TGFBR2 involves one or more of the insertion, deletion, or substitution of nucleotides, thereby incorporating them into the template nucleic acid. In some embodiments, modification of TGFBR2 involves the insertion of a donor nucleic acid into the target sequence. In some embodiments, modification of TGFBR2 is not transient.

[0259] In some implementations, the genetic modification results in a change in the nucleic acid sequence, which prevents the translation of the full-length protein having the amino acid sequence of the full-length protein before the genetic modification.

[0260] In some embodiments, gene modification results in changes to the nucleic acid sequence, leading to premature stop codons in the coding sequence of the full-length protein. In some embodiments, gene modification results in changes to the nucleic acid sequence, leading to changes in the splicing of pre-mRNA from genomic loci. In some embodiments, repression results in reduced cell surface expression of proteins from genes containing gene modifications.

[0261] 3. The efficacy of guide RNA The efficacy of the TGFBR2 guide RNA can be determined using techniques available in the art that assess the editing efficiency of the guide RNA and the surface expression of the TGFBR2 protein. In some embodiments, the reduction or elimination of the surface expression of the TGFBR2 protein can be determined by comparison with unmodified cells (or "relative to unmodified cells"). Engineered cells or cell populations can also be compared with unmodified cell populations.

[0262] "Unmodified cells" (or "multiple unmodified cells") refer to control cells (or multiple cells) of the same type used in the experiment or test, wherein the "unmodified" control cells have not yet been contacted with the TGFBR2 guide RNA. Therefore, unmodified cells (or multiple cells) can be cells that have not yet been contacted with the guide RNA, or cells that have been contacted with guide RNA that does not target TGFBR2.

[0263] In some embodiments, the efficacy of the TGFBR2 guide RNA is determined by measuring the surface expression level of the TGFBR2 protein. In some embodiments, the TGFBR2 protein level is measured by flow cytometry (e.g., using an antibody against TGFBR2). The surface expression of the TGFBR2 protein can be measured by flow cytometry as is commonly known in the art. Those skilled in the art will be familiar with techniques for measuring the surface expression of proteins such as TGFBR2 by flow cytometry. In some embodiments, the TGFBR2 protein level is measured indirectly by phosphorylated SMAD (pSMAD) signaling. Intracellular phosphorylated SMAD signaling levels are correlated with TGFBR2 protein levels. Those skilled in the art will be familiar with techniques for determining TGFBR2 protein levels by measuring phosphorylated SMAD signals. Exemplary measurements of the surface expression level of the TGFBR2 protein by flow cytometry are described in Examples 1-3. In some embodiments, as measured by flow cytometry, the cell population is enriched (e.g., by FACS or MACS) and at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, or 94% TGFBR2 negative relative to the unmodified cell population. In some embodiments, as measured by flow cytometry, the cell population is not enriched (e.g., by FACS or MACS) and at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, or 94% TGFBR2 negative relative to the unmodified cell population. In some embodiments, as measured by flow cytometry, at least 65% of the cell population is TGFBR2 negative relative to the unmodified cell population. In some embodiments, as measured by flow cytometry, at least 70% of the cell population is TGFBR2 negative relative to the unmodified cell population. In some embodiments, as measured by flow cytometry, at least 80% of the cell population is TGFBR2 negative relative to the unmodified cell population. In some embodiments, as measured by flow cytometry, at least 90% of the cell population is TGFBR2 negative relative to the unmodified cell population. In some embodiments, as measured by flow cytometry, at least 100% of the cell population is TGFBR2 negative relative to the unmodified cell population.

[0264] D. Methods and compositions for additional gene modification In some embodiments, multiplex gene editing can be performed in cells. In some embodiments, the method includes reducing or eliminating surface expression of the TGFBR2 protein, which includes genetic modification of the TGFBR2 gene, said genetic modification including contacting the cell with a composition comprising: the TGFBR2 guide RNA disclosed herein; and optionally an RNA-guided DNA binder or nucleic acid encoding the RNA-guided DNA binder, said method further including contacting with one or more compositions selected from: (a) a guide RNA that directs the RNA-guided DNA binder to the TGFBR2 gene; (b) a guide RNA that directs the RNA-guided DNA binder to a locus other than TGFBR2 in the cell genome; and (c) a donor nucleic acid for insertion into the cell genome.

[0265] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, and further has reduced or eliminated surface expression of one or more of MHC class II proteins, MHC-I proteins, TRAC, or TRBC relative to unmodified cells. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, and further has reduced or eliminated surface expression of HLA-A relative to unmodified cells. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, and further has reduced or eliminated surface expression of HLA-A (relative to unmodified cells) and surface expression of one or more of MHC class II proteins, TRAC, or TRBC (relative to unmodified cells). Such methods and compositions for reducing or eliminating the surface expression of one or more of MHC class II proteins, MHC-I proteins, TRAC, or TRBC are further described, for example, in International Publications WO 2020 / 081613, WO 2022 / 125982, WO 2022 / 140586, and WO 2022 / 140587 and International Applications PCT / US2023 / 068498 and PCT / US2023 / 068499, the contents of each of which are hereby incorporated in their entirety. For instance, a further detailed description of guide RNAs for reducing or eliminating TRBC and / or TRAC protein expression and for genetically modifying TRBC and / or TRAC is provided in International Publication WO 2020 / 081613, the entire contents of which are incorporated herein by reference. For example, a further detailed description of guide RNAs for reducing or eliminating HLA-A and / or CIITA protein expression and for genetic modification of HLA-A and / or CIITA is provided in International Publication No. WO 2022 / 125982, the entire contents of which are incorporated herein by reference. For example, a further detailed description of guide RNAs for reducing or eliminating HLA-A protein expression and for genetic modification of HLA-A is provided in International Publication No. WO 2022 / 140586, the entire contents of which are incorporated herein by reference. For example, a further detailed description of guide RNAs for reducing or eliminating HLA-A and / or CIITA protein expression and for genetic modification of HLA-A and / or CIITA is provided in International Publication No. WO 2022 / 140587, the entire contents of which are incorporated herein by reference.For example, a further detailed description of guide RNAs for reducing or eliminating HLA-A and / or HLA-B protein expression and for genetic modification of HLA-A and / or HLA-B is provided in International Application No. PCT / US2023 / 068498, the entire contents of which are incorporated herein by reference. Similarly, a further detailed description of guide RNAs for reducing or eliminating HLA-A, TRAC, TRBC, and / or CIITA protein expression and for genetic modification of HLA-A, TRAC, TRBC, and / or CIITA is provided in International Application No. PCT / US2023 / 068499, the entire contents of which are incorporated herein by reference.

[0266] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of the TGFBR2 protein relative to an unmodified cell, and containing a genetic modification of the TGFBR2 gene, wherein the genetic modification contains at least one nucleotide in any of the genomic coordinates shown in Table 2, and wherein the engineered cell further contains a genetic modification of one or more of the CIITA, HLA-A, HLA-B, TRAC, or TRBC genes.

[0267] In some embodiments, the methods and compositions include genetically modifying TGFBR2 using a gene editing system to reduce or eliminate surface expression of the TGFBR2 protein, and genetically modifying the insertion of exogenous nucleic acids encoding target receptors or other polypeptides (expressed or secreted on the cell surface) into the cell.

[0268] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of the TGFBR2 protein relative to an unmodified cell, and comprising a genetic modification of the TGFBR2 gene, wherein the genetic modification comprises at least one nucleotide within any of the genomic coordinates shown in Table 2, and wherein the engineered cell further comprises a foreign nucleic acid. In some embodiments, the engineered cell comprises a foreign nucleic acid encoding a targeting receptor expressed on the surface of the engineered cell. In some embodiments, the targeting receptor is a CAR or a universal CAR. In some embodiments, the targeting receptor is a TCR. In some embodiments, the targeting receptor is a WT1 TCR. In some embodiments, the targeting receptor is a ligand of a receptor. In some embodiments, the targeting receptor is a heterozygous CAR / TCR. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain) and a subunit of the TCR. In some embodiments, the targeting receptor is a cytokine receptor. In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a B cell receptor (BCR).

[0269] In some embodiments, the engineered cells also contain exogenous nucleic acids encoding polypeptides (i.e., soluble polypeptides) secreted by the engineered cells. In some embodiments, the exogenous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the secreted polypeptide is an antibody. In some embodiments, the secreted polypeptide is an enzyme. In some embodiments, the exogenous nucleic acid encodes an antibody that encodes a cytokine. In some embodiments, the exogenous nucleic acid encodes a chemokine. In some embodiments, the exogenous nucleic acid encodes a fusion protein.

[0270] In some embodiments, an engineered cell is provided that, compared to unmodified cells, has reduced or eliminated surface expression of the TGFBR2 protein, and further has reduced or eliminated surface expression of MHC class II proteins. In some embodiments, the engineered cell has genetic modifications in its genes that reduce or eliminate the surface expression of MHC class II proteins.

[0271] In some embodiments, methods are provided for reducing or eliminating the surface expression of TGFBR2 by genetic modification as disclosed herein, wherein said methods and compositions further provide for reducing or eliminating the surface expression of MHC class II proteins relative to unmodified cells. In some embodiments, the expression of MHC class II proteins is reduced or eliminated by contacting cells with CIITA guide RNA.

[0272] MHC class II expression is influenced by a variety of proteins. In some embodiments, MHC class II protein expression is reduced or eliminated by genetic modification of genes selected from the following: CIITA, HLA-DR, HLA-DQ, HLA-DP, RFX5, RFXB / ANK, RFXAP, CREB, NF-YA, NF-YB, and NF-YC. In some embodiments, MHC class II protein expression is reduced or eliminated by genetic modification of the CIITA gene.

[0273] In some embodiments, the engineered cells have a gene modification in the CIITA gene. In some embodiments, the engineered cells have a gene modification in the HLA-DR gene. In some embodiments, the engineered cells have a gene modification in the HLA-DQ gene. In some embodiments, the engineered cells have a gene modification in the HLA-DP gene. In some embodiments, the engineered cells have a gene modification in the RFX gene. In some embodiments, the engineered cells have a gene modification in the CREB gene. In some embodiments, the engineered cells have a gene modification in the nuclear factor (NF)-γ gene.

[0274] In some embodiments, a method is provided for manufacturing engineered cells having reduced or eliminated expression of the TGFBR2 protein relative to unmodified cells, the method further comprising reducing or eliminating surface expression of MHC class II proteins in the cells relative to unmodified cells. In some embodiments, the method includes contacting the cells with CIITA guide RNA.

[0275] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, and further has reduced or eliminated surface expression of TRBC protein. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, and further has reduced or eliminated surface expression of TRBC protein.

[0276] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, and further has reduced or eliminated surface expression of HLA-A protein. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, and further has reduced or eliminated surface expression of HLA-B protein. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TGFBR2 protein relative to unmodified cells, and further has reduced or eliminated surface expression of both HLA-A and HLA-B proteins.

[0277] In some embodiments, the engineered cells contain gene modifications in one or more of the HLA-A, HLA-B, TRAC, TRBC, or CIITA genes. In some embodiments, the gene modifications in the HLA-A gene are located within the HLA-A target genomic coordinates shown in Tables 10A-10B (e.g., chr6:29942891-29942915, chr6:29942609-29942633, or chr6:29942864-29942884). In some embodiments, the gene modifications in the HLA-B gene are located within the HLA-B target genomic coordinates shown in Tables 10A-10B (e.g., chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229). In some embodiments, gene modifications in the TRAC gene are located within the TRAC target genome coordinates shown in Tables 10A-10B (e.g., chr14:22547524-22547544, chr14:22550574-22550598, or chr14:22550544-22550568). In some embodiments, gene modifications in the CIITA gene are located within the CIITA target genome coordinates shown in Tables 10A-10B (e.g., chr16:10906643-10906667, chr16:10907504-10907528, or chr16:10906853-10906873). In some implementations, the genetic modifications in the TRBC gene are located within the TRBC target genome coordinates shown in Tables 10A-10B (e.g., chr7:142792690-142792714 or chr7:142792047-142792067).

[0278] In some embodiments, the engineered cells contain gene modifications of one or more of the HLA-A, HLA-B, TRAC, TRBC, or CIITA genes. In some embodiments, the gene modification in the HLA-A gene contains at least one nucleotide within a genomic coordinate targeted by an HLA-A guide RNA, said guide RNA containing a guide sequence selected from SEQ ID NO: 403, 404, and 412. In some embodiments, the gene modification in the HLA-B gene contains at least one nucleotide within a genomic coordinate targeted by an HLA-B guide RNA, said guide RNA containing a guide sequence selected from SEQ ID NO: 406, 405, and 407. In some embodiments, the gene modification in the TRAC gene contains at least one nucleotide within a genomic coordinate targeted by a TRAC guide RNA, said guide RNA containing a guide sequence selected from SEQ ID NO: 413, 408, and 409. In some embodiments, the gene modification in the CIITA gene contains at least one nucleotide within a genomic coordinate targeted by a CIITA guide RNA, said guide RNA containing a guide sequence selected from SEQ ID NO: 402, 401, and 411. In some embodiments, the genetic modification in the TRBC includes at least one nucleotide within a genomic coordinate targeted by the TRBC guide RNA, said guide RNA comprising the guide sequence of SEQ ID NO: 410 or 414.

[0279] In some embodiments, in any of the methods and compositions disclosed herein, the HLA-A guide RNA is an HLA-A guide RNA comprising guide sequences disclosed in Tables 10A-10B, such as guide sequences selected from SEQ ID NO: 403, 404, and 412. In some embodiments, in any of the methods and compositions disclosed herein, the HLA-B guide RNA is an HLA-B guide RNA comprising guide sequences disclosed in Tables 10A-10B, such as guide sequences selected from SEQ ID NO: 406, 405, and 407. In some embodiments, in any of the methods and compositions disclosed herein, the TRAC guide RNA is a TRAC guide RNA comprising guide sequences disclosed in Tables 10A-10B, such as guide sequences selected from SEQ ID NO: 413, 408, and 409. In some embodiments, in any of the methods and compositions disclosed herein, the CIITA guide RNA is a CIITA guide RNA comprising a guide sequence disclosed in Tables 10A-10B, such as a guide sequence selected from SEQ ID NO: 402, 401, and 411. In some embodiments, in any of the methods and compositions disclosed herein, the TRBC guide RNA is a TRBC guide RNA comprising a guide sequence disclosed in Tables 10A-10B, such as a guide sequence selected from SEQ ID NO: 410 and 414.

[0280] In some embodiments, in any of the methods and compositions disclosed herein, the HLA-A guide RNA disclosed herein is a single guide RNA comprising the sequences disclosed in Tables 10A-10B. In some embodiments, in any of the methods and compositions disclosed herein, the HLA-B guide RNA disclosed herein is a single guide RNA comprising the sequences disclosed in Tables 10A-10B. In some embodiments, in any of the methods and compositions disclosed herein, the CIITA guide RNA disclosed herein is a single guide RNA comprising the sequences disclosed in Tables 10A-10B. In some embodiments, in any of the methods and compositions disclosed herein, the TRAC guide RNA disclosed herein is a single guide RNA comprising the sequences disclosed in Tables 10A-10B. In some embodiments, in any of the methods and compositions disclosed herein, the TRBC guide RNA disclosed herein is a single guide RNA comprising the sequences disclosed in Tables 10A-10B.

[0281] In some embodiments, an engineered cell is provided having gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, and / or the TGFBR2 gene, wherein the gene modification in the HLA-A gene is located within genomic coordinates chr6:29942891-29942915; the gene modification in the HLA-B gene is located within genomic coordinates chr6:31355222-31355246; the gene modification in the TRAC gene is located within genomic coordinates chr14:22547524-22547544; the gene modification in the CIITA gene is located within genomic coordinates chr16:10906643-10906667; and the gene modification in the TGFBR2 gene is located within genomic coordinates chr3:30674205-30674229.

[0282] In some embodiments, in the engineered cells, cell populations, pharmaceutical compositions, or methods disclosed herein, the engineered cells comprise (i) gene modifications within genomic coordinates targeted by an HLA-A guide RNA containing the guide sequence of SEQ ID NO: 403; (ii) gene modifications within genomic coordinates targeted by an HLA-B guide RNA containing the guide sequence of SEQ ID NO: 406; (iii) gene modifications within genomic coordinates targeted by a TRAC guide RNA containing the guide sequence of SEQ ID NO: 413; (iv) gene modifications within genomic coordinates targeted by a CIITA guide RNA containing the guide sequence of SEQ ID NO: 402; and (v) gene modifications within genomic coordinates targeted by a TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 1.

[0283] In some embodiments, in the engineered cells, cell populations, pharmaceutical compositions, or methods disclosed herein, the engineered cells provided herein are generated by a genome editing system comprising one or more of the following, or the compositions provided herein comprise one or more of the following: HLA-A guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 446; HLA-B guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 452; HLA-B guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 444; HLA-C guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 444; HLA-B ...A guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 464 TRAC guide RNA having a guide sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity, and TGFBR2 guide RNA containing a guide sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 201.

[0284] In some embodiments, the engineered cells further include gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, and the CIITA gene. In some embodiments, the engineered cells also include gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, and the TGFβR2 gene.

[0285] In some implementations, the engineered cells further include: i. gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene; ii. gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene; iii. gene modifications within the genomic coordinates chr14:22547524-22547544 of the TRAC gene; and iv. gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene. In some implementations, the engineered cells include: i. gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene; ii. gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene; iii. gene modifications within the genomic coordinates chr14:22547524-22547544 of the TRAC gene; iv. gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene; and v. gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFβR2 gene.

[0286] In some embodiments, this document provides an engineered human cell comprising gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFβR2 gene, and gene modifications within the genomic coordinates chr14:22547524-22547544 of the TRAC gene.

[0287] E. Exogenous nucleic acid knock-in In some embodiments, this disclosure provides methods and compositions for reducing or eliminating surface expression of the TGFBR2 protein by genetically modifying TGFBR2 as disclosed herein, wherein said methods and compositions further provide expression of proteins encoded by exogenous nucleic acids (e.g., antibodies, chimeric antigen receptors (CARs), T-cell receptors (TCRs), cytokines or cytokine receptors, chemokines or chemokine receptors, enzymes, fusion proteins, or other types of cell surface-binding peptides or soluble peptides). In some embodiments, the exogenous nucleic acid encodes a protein expressed on the cell surface. For example, in some embodiments, the exogenous nucleic acid encodes a targeting receptor expressed on the cell surface (further described herein). In some embodiments, the genetically modified cell can act as a “cell factory” for expressing secretory peptides encoded by the exogenous nucleic acid, for example, as a source of continuous in vivo peptide production (as further described herein). In some embodiments, the cell is an allogeneic cell.

[0288] In some embodiments, the method includes reducing the surface expression of the TGFBR2 protein, which includes genetically modifying the TGFBR2 gene, said genetic modification including contacting cells with a composition containing the TGFBR2 guide RNA disclosed herein, and said method further includes contacting cells with exogenous nucleic acids.

[0289] In some embodiments, the method includes reducing or eliminating surface expression of the TGFBR2 protein, which includes genetically modifying cells with one or more compositions comprising a TGFBR2 guide RNA as disclosed herein, an exogenous nucleic acid encoding a polypeptide (e.g., a target receptor), and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0290] In some embodiments, the method includes reducing or eliminating the surface expression of TGFBR2 protein and MHC class II protein, which includes genetically modifying cells with one or more compositions comprising TGFBR2 guide RNA, CIITA guide RNA, exogenous nucleic acid encoding a polypeptide (e.g., a target receptor) as disclosed herein, and RNA-guided DNA binders or nucleic acids encoding RNA-guided DNA binders.

[0291] In some embodiments, the exogenous nucleic acid encodes a polypeptide expressed on the cell surface. In some embodiments, the exogenous nucleic acid encodes a soluble polypeptide. As used herein, a "soluble" polypeptide refers to a polypeptide secreted by cells. In some embodiments, the soluble polypeptide is a therapeutic polypeptide. In some embodiments, the soluble polypeptide is an antibody. In some embodiments, the soluble polypeptide is an enzyme. In some embodiments, the soluble polypeptide is a cytokine. In some embodiments, the soluble polypeptide is a chemokine. In some embodiments, the soluble polypeptide is a fusion protein.

[0292] In some embodiments, the exogenous nucleic acid encodes an antibody. In some embodiments, the exogenous nucleic acid encodes an antibody fragment (e.g., Fab, Fab2). In some embodiments, the exogenous nucleic acid encodes a full-length antibody. In some embodiments, the exogenous nucleic acid encodes a single-chain antibody (e.g., scFv). In some embodiments, the antibody is IgG, IgM, IgD, IgA, or IgE. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the heavy chain constant region contains a mutation known to reduce effector function. In some embodiments, the heavy chain constant region contains a mutation known to enhance effector function. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a single-domain antibody (e.g., a VH domain-only antibody).

[0293] In some embodiments, the exogenous nucleic acid encodes a neutralizing antibody. A neutralizing antibody neutralizes the activity of its target antigen. In some embodiments, the antibody is a neutralizing antibody against a viral antigen. In some embodiments, the antibody neutralizes the target viral antigen, thereby blocking the virus's ability to infect cells. In some embodiments, the neutralizing activity of the antibody can be measured using a cell-based neutralization assay. The specific cells and readings will depend on the target antigen of the neutralizing antibody. The half-maximum effective concentration (CMC) of the antibody... 50 It can be measured in cell-based neutralization assays, where lower EC50 values ​​are... 50 Indicates a strong neutralizing antibody.

[0294] In some implementations, the exogenous nucleic acid encodes an antibody that binds to an antigen associated with a disease or condition (see, for example, the diseases and conditions described in Section X).

[0295] In some embodiments, the exogenous nucleic acid encodes a polypeptide (i.e., a cell surface binding protein) expressed on the cell surface. In some embodiments, the exogenous nucleic acid encodes a targeting receptor. A “targeting receptor” is a receptor present on the surface of a cell (e.g., a T cell) to allow the cell to bind to a target site (e.g., a specific cell or tissue in an organism). In some embodiments, the targeting receptor is a CAR. In some embodiments, the targeting receptor is a universal CAR (UniCAR). In some embodiments, the targeting receptor is an aplastic proliferation ligand (APRIL). In some embodiments, the targeting receptor is a TCR. In some embodiments, the targeting receptor is a TruC. In some embodiments, the targeting receptor is a B cell receptor (BCR) (e.g., expressed on B cells). In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a cytokine receptor.

[0296] In some embodiments, the target receptor includes a chimeric antigen receptor (CAR), a T-cell receptor (TCR), and a receptor for a cell surface molecule, said receptor being operatively linked via at least one transmembrane domain in an internal signaling domain capable of activating T cells upon binding to an extracellular receptor portion. In some embodiments, CAR refers to an extracellular antigen recognition domain, such as scFv, VHH, or nanobodies; which is operatively linked to an intracellular signaling domain that activates T cells upon binding to an antigen. A CAR consists of four regions: an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular T-cell signaling domain. Such receptors are well known in the art (see, for example, WO2020092057, WO2019191114, WO2019147805, WO2018208837). This also covers universal CARs (UniCARs) for recognizing a variety of antigens (see, for example, EP 2 990 416A1) and reverse universal CARs (RevCARs) that facilitate the binding of immune cells to target cells via adaptor molecules (see, for example, WO2019238722). CARs can target any antigen that can generate antibodies and are typically targeted at molecules displayed on the surface of the cells or tissues to be targeted. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain) and a subunit of the TCR (e.g., TruC). (See Baeuerle et al., Nature Communications 2087 (2019)). In some embodiments, the exogenous nucleic acid encodes a TCR. In some embodiments, the exogenous nucleic acid encodes a gene-modified TCR. In some embodiments, the exogenous nucleic acid encodes a gene-modified TCR that is specific to a polypeptide expressed by cancer cells. In some embodiments, the exogenous nucleic acid encodes a targeting receptor specifically targeting the Wilms' tumor gene (WT1) antigen. In some embodiments, the exogenous nucleic acid encodes a WT1-specific TCR (see, for example, WO2020 / 081613A1).

[0297] In some embodiments, exogenous nucleic acids are inserted into the genome of the target cell. In some embodiments, exogenous nucleic acids are integrated into the genome of the target cell. In some embodiments, exogenous nucleic acids are integrated into the genome of the target cell via homologous recombination (HR). In some embodiments, exogenous nucleic acids are integrated into the genome of the target cell via blunt-end insertion. In some embodiments, exogenous nucleic acids are integrated into the genome of the target cell via non-homologous end joining. In some embodiments, exogenous nucleic acids are integrated into a safe harbor locus in the cell genome. In some embodiments, exogenous nucleic acids are integrated into one of the TRAC locus, B2M locus, AAVS1 locus, or CIITA locus. In some embodiments, the lipid-nucleic acid assembly composition is lipid nanoparticles (LNPs).

[0298] In some embodiments, the method produces a composition comprising engineered cells having reduced or eliminated surface expression of the TGFBR2 protein and containing exogenous nucleic acids. In some embodiments, the method produces a composition comprising engineered cells having reduced or eliminated surface expression of the TGFBR2 protein and secreting or expressing a polypeptide encoded by an exogenous nucleic acid integrated into the cell's genome. In some embodiments, the method produces a composition comprising engineered cells having reduced or eliminated surface expression of the TGFBR2 protein, or reduced or eliminated TGFBR2 levels in the cell nucleus, and having reduced or eliminated surface expression of MHC class II proteins, and secreting or expressing a polypeptide encoded by an exogenous nucleic acid integrated into the cell's genome. In some embodiments, the engineered cells induce a weakened response from CD4+ T cells or CD8+ T cells.

[0299] In some embodiments, an allogeneic cell is provided, wherein the cell has reduced or eliminated surface expression of MHC class II and TGFBR2 proteins, wherein the cell contains modifications in the TGFBR2 gene as disclosed herein, wherein the cell contains modifications in the CIITA gene, and wherein the cell further contains exogenous nucleic acids encoding polypeptides (e.g., targeting receptors).

[0300] In some embodiments, this disclosure provides methods for reducing or eliminating the surface expression of the TGFBR2 protein by genetically modifying it, as disclosed herein, wherein said methods further provide for reducing the expression of one or more additional target genes (e.g., HLA-A, HLA-B, CIITA, TRAC, TRBC). In some embodiments, the additional genetic modification provides further advantages for the use of the genetically modified cells in adoptive cell transfer applications.

[0301] In some embodiments, the method includes reducing or eliminating the surface expression of the TGFBR2 protein, which includes genetically modifying cells with one or more compositions comprising a TGFBR2 guide RNA as disclosed herein; a CIITA guide RNA; a foreign nucleic acid encoding a polypeptide (e.g., a target receptor); a guide RNA that directs an RNA-guided DNA binder to a target sequence located in another gene, thereby reducing or eliminating the expression of said other gene; and an RNA-guided DNA binder or nucleic acid encoding an RNA-guided DNA binder. In some embodiments, an additional target gene is TRAC. In some embodiments, an additional target gene is TRBC.

[0302] In some embodiments, the methods disclosed herein further include contacting cells with a DNA-dependent protein kinase inhibitor (DNAPKi), optionally wherein said DNAPKi is compound 1 or "DNAPKI compound 1": 9-(4,4-difluorocyclohexyl)-7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,9-dihydro-8H-purine-8-one, also described as: .

[0303] In some embodiments, an engineered cell is provided, the engineered cell having gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, and / or the TGFBR2 gene, wherein the gene modification in the HLA-A gene is located within genomic coordinates chr6:29942891-29942915; wherein the gene modification in the HLA-B gene is located within genomic coordinates chr6:31355222-31355246; wherein... The gene modification in the TRAC gene is located within genomic coordinates chr14:22547524-22547544; the gene modification in the CIITA gene is located within genomic coordinates chr16:10906643-10906667; and the gene modification in the TGFBR2 gene is located within genomic coordinates chr3:30674205-30674229. The cell contains exogenous nucleic acid encoding a target receptor expressed on the surface of the engineered cell, wherein the target receptor is a TCR or CAR.

[0304] In some embodiments, in the engineered cells, cell populations, pharmaceutical compositions, or methods disclosed herein, the engineered cells comprise (i) gene modifications within genomic coordinates targeted by an HLA-A guide RNA containing the guide sequence of SEQ ID NO: 403; (ii) gene modifications within genomic coordinates targeted by an HLA-B guide RNA containing the guide sequence of SEQ ID NO: 406; (iii) gene modifications within genomic coordinates targeted by a TRAC guide RNA containing the guide sequence of SEQ ID NO: 413; (iv) gene modifications within genomic coordinates targeted by a CIITA guide RNA containing the guide sequence of SEQ ID NO: 402; and (v) gene modifications within genomic coordinates targeted by a TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 1, and wherein the cells comprise exogenous nucleic acids encoding a target receptor expressed on the surface of the engineered cells, wherein the target receptor is a TCR or a CAR.

[0305] In some embodiments, in the engineered cells, cell populations, pharmaceutical compositions, or methods disclosed herein, the engineered cells provided herein are generated by a genome editing system comprising one or more of the following, or the compositions provided herein comprise one or more of the following: HLA-A guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 446; HLA-B guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 452; HLA-B guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 444; HLA-C guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 444; HLA-B ...A guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 464 includes a TRAC guide RNA having a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the guide sequence of SEQ ID NO: 201, and a TGFBR2 guide RNA comprising a guide sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the guide sequence of SEQ ID NO: 201, wherein the composition further comprises an exogenous nucleic acid encoding a target receptor expressed on the surface of the engineered cell, wherein the target receptor is a TCR or a CAR.

[0306] F. Exemplary Genome Editing Systems The engineered cells disclosed herein can be created using a variety of suitable gene editing systems, including but not limited to CRISPR / Cas systems; zinc finger nuclease (ZFN) systems; and transcription activator-like effector nuclease (TALEN) systems. Generally, gene editing systems involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks (e.g., single-strand breaks or SSBs) in a target DNA sequence. Cleavage or nicks can occur via the use of specific nucleases (such as engineered ZFNs, TALENs) or by using CRISPR / Cas systems with engineered guide RNAs to guide specific cleavage or nicks of the target DNA sequence. Furthermore, targeted nucleases based on Argonaute systems (e.g., from *T. thermophilus*, referred to as 'TtAgo', see Swarts et al. (2014) Nature 507(7491): 258-261) are being developed, which may also have potential for use in gene editing and gene therapy.

[0307] In some implementations, the gene editing system is a TALEN system. A transcription activator-like effector nuclease (TALEN) is a restriction enzyme that can be engineered to cleave specific DNA sequences. This nuclease is prepared by fusing a TAL effector DNA-binding domain with a DNA-cleaving domain (a nuclease that cuts the DNA strand). Transcription activator-like effectors (TALEs) can be engineered to bind to desired DNA sequences, thereby promoting DNA cleavage at a specific location (see, for example, Boch, 2011, Nature Biotech). Restriction enzymes can be introduced into cells for gene editing or for in situ gene editing; this technique is known as gene editing using engineered nucleases. Such methods and compositions used therein are known in the art. See, for example, WO2019147805, WO2014040370, and WO2018073393, the contents of which are hereby incorporated in their entirety.

[0308] In some implementations, the gene editing system is a zinc finger system. Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by fusing a zinc finger DNA-binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, enabling the zinc finger nuclease to target unique sequences within a complex genome. The non-specific cleavage domain of the type II restriction endonuclease FokI is commonly used as the cleavage domain in ZFNs. Cleavage is repaired by endogenous DNA repair mechanisms, allowing the ZFN to precisely alter the genome of higher organisms. Such methods and compositions used therein are known in the art. See, for example, WO2011091324, the contents of which are hereby incorporated in their entirety.

[0309] In some embodiments, the gene editing system is a CRISPR / Cas system, comprising, for example, a guide sequence and an RNA-guided DNA binder, and a CRISPR guide RNA further described herein. In some embodiments, the gene editing system comprises a base editor containing a deaminase and an RNA-guided nicking enzyme. In some embodiments, the gene editing system comprises a base editor containing a cytidine deaminase and an RNA-guided nicking enzyme. In some embodiments, the gene editing system comprises a DNA polymerase. Further descriptions of gene editing system methods and compositions used therein are known in the art. See, for example, WO2019 / 067910, WO2021 / 188840A1, WO2019 / 051097 and PCT / US2021 / 062922, filed December 10, 2021, and U.S. Provisional Application No. 63 / 275,425, filed November 3, 2021, the contents of each of which are hereby incorporated in their entirety. Exemplary nucleotide and polypeptide sequences of the gene editing systems disclosed herein are provided in Table 10 below. Methods for identifying alternative nucleotide sequences (including naturally occurring variants of the alternatives) encoding the polypeptide sequences provided herein are known in the art. Also covered are sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with either the nucleic acid sequence or the nucleic acid sequence encoding the amino acid sequence provided herein.

[0310] G. CRISPR guide RNA This article provides guide sequences that can be used to modify target sequences, for example, by using guide RNA containing the disclosed guide sequences with an RNA-guided DNA binder (e.g., a CRISPR / Cas system).

[0311] In some aspects, a guide RNA (gRNA) comprising a guide region and a conserved region is provided, wherein: A. the guide region comprises a nucleic acid sequence having at least 80%, 85%, preferably 90% or 95% identity or complementarity to 24 consecutive nucleotides of any of the guide sequences (SEQ ID NO: 1-49) in Table 2.

[0312] In some embodiments, the conserved region comprises one or more of the following: (a) a shortened repeat / anti-repeat region, wherein the shortened repeat / anti-repeat region is missing 2-24 nucleotides relative to SEQ ID NO: 700, wherein (i) one or more of nucleotides 37-48 and 53-64 are missing relative to SEQ ID NO: 700 and optionally one or more of nucleotides 37-64 are substituted relative to SEQ ID NO: 700; and (ii) nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b) a shortened hairpin 1 region, wherein the shortened hairpin 1 is missing 2-10 nucleotides, optionally 2-8 nucleotides, relative to SEQ ID NO: 700, wherein (i) one or more of nucleotides 82-86 and 91-95 are missing relative to SEQ ID NO: 700 and optionally one or more of positions 82-96 are missing relative to SEQ ID NO: 700. 700 is substituted; and (ii) nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (c) a shortened hairpin 2 region, wherein the shortened hairpin 2 is missing 2-18 nucleotides relative to SEQ ID NO: 700, optionally 2-16 nucleotides, wherein (i) one or more of nucleotides 113-121 and 126-134 are missing relative to SEQ ID NO: 700 and optionally one or more of nucleotides 113-134 are substituted relative to SEQ ID NO: 700; and (ii) nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides; wherein one or two of nucleotides 144-145 are optionally missing relative to SEQ ID NO: 700; optionally, wherein at least 10 nucleotides are modified nucleotides.

[0313] In some implementations, the conserved region contains nucleotide sequences selected from Tables 4-7.

[0314] In some implementations, the guide RNA contains at least one end modification.

[0315] In some implementations, the modification includes a 5' end modification.

[0316] In some implementations, the modification includes a 3' end modification.

[0317] In some implementations, the guide RNA includes modifications in the hairpin region.

[0318] In some implementations, the embellishments in the hairpin area are also end embellishments.

[0319] In some implementations, the nucleotide is modified to include a 2'-O-methyl (2'-O-Me) modification.

[0320] In some implementations, the modification involves the phosphate thioester (PS) bond between nucleotides.

[0321] In some implementations, the nucleotide modified to include a 2'-O-methyl (2'-O-Me) modification is linked to a 3' adjacent nucleotide via a phosphate thioester (PS) bond.

[0322] In some implementations, the nucleotides are modified to include 2'-fluorine (2'F) modification.

[0323] In some embodiments, the 5' end modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide at positions 1-3 of the 5' end of the guide sequence, which is linked to the 3' adjacent nucleotide by a phosphate thioester (PS) bond.

[0324] In some embodiments, the conserved region comprises a modified nucleotide sequence selected from the modified conserved region Nme guide RNA motifs in Table 6, and wherein the conserved region is at the 3' of the guide region.

[0325] In some implementations, the guide RNA comprises a nucleotide sequence selected from any of the guide sequences in Table 2.

[0326] In some implementations, each nucleotide is any natural or non-natural nucleotide.

[0327] In some embodiments, the guide RNA is modified according to a pattern selected from SEQ ID NO: 710-732, wherein N is the guide sequence described herein, and N, A, C, G and U are ribonucleotides (2'-OH), wherein "m" indicates 2'-O-Me modification, "f" indicates 2'-fluorine modification, and "*" indicates phosphate thioester linkage between nucleotides.

[0328] In some aspects, this document provides a composition comprising the guide RNA described herein.

[0329] Guide sequences targeting sites adjacent to a suitable PAM (e.g., NmeCas9 PAM) (e.g., as shown in Table 1) may also contain additional nucleotides to form crRNA or crRNA that conjugates with trRNA to form sgRNA, for example, exemplary nucleotide sequences following the guide sequence at its 3' end, as provided in Tables 4-7. Exemplary NmeCas9 sgRNA part and position numbering schemes (including both guide sequences and scaffold sequences) are listed in Table 8 below.

[0330] In some implementations, SEQ ID NO: 700 (“Exemplary NmeCas9 sgRNA-1”) is used as an example, and Exemplary NmeCas9 sgRNA-1 includes: A. A guide RNA (gRNA) comprising a guide region and a conserved region, the conserved region comprising one or more of the following: (a) A shortened repeat / anti-repeat region, wherein the shortened repeat / anti-repeat region is missing 2-24 nucleotides, wherein (i) Relative to SEQ ID NO: 700, one or more of nucleotides 37-48 and 53-64 are deleted, and optionally one or more of nucleotides 37-64 are substituted; and (ii) Nucleotide 36 is composed of at least two nucleotides linked to nucleotide 65; or (b) A shortened hairpin 1 region, wherein the shortened hairpin 1 is missing 2-10, optionally 2-8 nucleotides, wherein (i) Relative to SEQ ID NO: 700, one or more of nucleotides 82-86 and 91-95 are deleted, and optionally one or more of positions 82-96 are substituted; and (ii) Nucleotide 81 is linked to nucleotide 96 by at least four nucleotides; or (c) The shortened hairpin 2 region, wherein the shortened hairpin 2 lacks 2-18, optionally 2-16 nucleotides, wherein (i) Relative to SEQ ID NO: 700, one or more of nucleotides 113-121 and 126-134 are deleted, and optionally one or more of nucleotides 113-134 are substituted; and (ii) Nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides; One or both of nucleotides 144-145 are optionally deleted relative to SEQ ID NO: 700; Optionally, at least 10 nucleotides are modified nucleotides.

[0331] Exemplary unmodified conserved nucleotide sequences (also known as scaffold sequences) are shown in Table 4. #mer refers to the length of the sgRNA when the 24-nucleotide guide sequence is included at the 5' of the scaffold sequence provided in Table 4.

[0332] Table 4: Exemplary unmodified Nme guide RNA conserved region nucleotide sequences In some implementations, the guide RNA comprises a nucleotide sequence selected from unmodified Nme guide RNA sequences in Table 2, wherein N 20-25 All of them are any of the guide sequences disclosed in Table 2. In some embodiments, each nucleotide in the unmodified Nme guide RNA sequence in Table 5 is any natural or non-natural nucleotide.

[0333] Table 5: Exemplary unmodified Nme guide RNA nucleotide sequences In the case of sgRNA, the modified guide sequence can be integrated into one of the following exemplary modified conserved motifs (Table 6). #mer refers to the length of the sgRNA when the 24-nucleotide guide sequence (modified or unmodified) is included at the 5' of the scaffold sequence provided in Table 6: Table 6: Conserved regions of exemplary modified Nme guide RNAs Where “m” indicates 2'-O-Me modification, and “*” indicates phosphate thioester bonding between nucleotides, and in the case of modified sequences, no modification indicates RNA (2'-OH) and phosphate thioester bonding.

[0334] The guide sequence is located at the 5' end of the conserved portion of the guide RNA. In some embodiments, the guide sequence is 20-25 nucleotides long, preferably 22-24 nucleotides. In some embodiments, the guide sequence includes one or more chemical modifications, such as modifications at nucleotides 1, 2, and 3 at the 5' end of the guide RNA, optionally modifications at all nucleotides 1, 2, and 3. In some embodiments, the modification includes a 2'-O-Me modification.

[0335] In some embodiments, the modification includes a 2'-O-Me modification and a phosphate thioester bond to a 3' nucleotide, such as (mN*)3(N). 17-22 Preferred (mN*)3(N) 21 , where (N) 21 Each nucleotide in the portion is either modified or unmodified independently.

[0336] In some embodiments, N as a whole constitutes a guide sequence comprising: (A) a sequence having at least 80%, 85%, preferably at least 90% or 95%, or 100% identity or complementarity to the 24 consecutive nucleotides of the guide sequences disclosed in Table 2. For example, N may be substituted with any of the guide sequences disclosed in Table 2 herein. In some embodiments, when N as a whole constitutes (N) 20-25 When the guide sequence is within (N), 20-25Each N can be modified independently, for example, with 2'-OMe modification, and optionally further modified with PS modification, particularly at the 1st, 2nd, or 3rd terminal nucleotide. In some embodiments, (N)20-25 has the following sequence and modification pattern: mN*mN*mN*mNmNmNNNmNmNNmNNNNmNNNNmNNNN.

[0337] In some embodiments, the sgRNA comprises any of the modification patterns shown herein, wherein N is any natural or non-natural nucleotide, and wherein N as a whole constitutes the guide sequence disclosed in Table 2. In some embodiments, the modified sgRNA comprises the sequences shown in Table 7.

[0338] Table 7: Exemplary modified Nme guide RNA sequences Where “m” indicates 2'-O-Me modification, and “*” indicates phosphate thioester bonding between nucleotides, and in the case of modified sequences, no modification indicates RNA (2'-OH) and phosphate thioester bonding.

[0339] In some embodiments, the sgRNA (such as sgRNA containing NmeCas9 sgRNA-1) includes a 3' tail, for example, a 3' tail of 1, 2, 3, 4 or more nucleotides. In some embodiments, the tail includes one or more modified nucleotides. In some embodiments, the modified nucleotide is selected from 2'-O-methyl (2'-OMe) modified nucleotides, 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, 2'-deoxy (2'H-) modified nucleotides, baseless nucleotides, locked nucleic acid (LNA) nucleotides, unlocked nucleic acid (UNA) nucleotides, phosphate thioester (PS) bonds between nucleotides, and terminally reverse baseless nucleotides; or combinations thereof. In some embodiments, the modified nucleotide includes a 2'-OMe modified nucleotide. In some embodiments, the modified nucleotide includes a PS bond between nucleotides. In some embodiments, the modified nucleotide includes a 2'-OMe modified nucleotide and a PS bond between nucleotides.

[0340] In some embodiments, the hairpin region includes one or more modified nucleotides. In some embodiments, the modified nucleotides are selected from 2'-O-methyl (2'-OMe) modified nucleotides, 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, phosphate thioester (PS) bonds between nucleotides, and reverse-base-free nucleotides; or combinations thereof. In some embodiments, the modified nucleotides include 2'-OMe modified nucleotides.

[0341] In some embodiments, the upper stem region includes one or more modified nucleotides. In some embodiments, the modified nucleotides are selected from 2'-O-methyl (2'-OMe) modified nucleotides, 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, phosphate thioester (PS) bonds between nucleotides, and reverse-base-free nucleotides; or combinations thereof. In some embodiments, the modified nucleotides include 2'-OMe modified nucleotides.

[0342] In some embodiments, the exemplary NmeCas9 sgRNA-1 comprises one or more YA dinucleotides, where Y is pyrimidine, and the YA dinucleotide includes a modified nucleotide. In some embodiments, the modified nucleotide is selected from 2'-O-methyl (2'-OMe) modified nucleotides, 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, phosphate thioester (PS) bonds between nucleotides, and reverse-base-free modified nucleotides, or combinations thereof. In some embodiments, the modified nucleotide includes a 2'-OMe modified nucleotide.

[0343] In some embodiments, the exemplary NmeCas9 sgRNA-1 comprises one or more YA dinucleotides, where Y is pyrimidine, and the YA dinucleotide includes a sequence-substituted nucleotide in which pyrimidine replaces a purine. In some embodiments, when pyrimidine forms a Watson-Crick base pair in a single guide, the sequence-substituted pyrimidine nucleotide is replaced with a Watson-Crick-based nucleotide to maintain the Watson-Crick base pairing.

[0344] gRNA containing linkers In some implementations, the gRNA contains one or more internal linkers. As used herein, "internal linker" describes a non-nucleotide segment of two nucleotides within the gRNA that binds. If the gRNA contains a spacer region, the internal linker is located outside the spacer region (e.g., within the scaffold or conserved region of the gRNA). For V-shaped guides, it should be understood that the final hairpin is the only hairpin in the structure (i.e., the repeat-anti-repeat region). The length of the internal linker can depend, for example, on the number of nucleotides replaced by the linker and the linker's position within the gRNA. Internal linkers and their use in the gRNA case are provided in WO2022261292.

[0345] The gRNAs disclosed herein may contain internal linkers. Generally, any internal linker compatible with the function of the gRNA can be used. A degree of flexibility in the linker may be desirable. In some embodiments, the internal linker contains at least two, three, four, five, six, or more on-pathway single bonds. A linker is considered on-pathway if it is part of the shortest path between two nucleotides connected to the linker at the 5' and 3' positions.

[0346] As used herein, the length of an internal linker can be defined by its bridging length. As used herein, the “bridging length” of an internal linker refers to the distance or number of atoms in the shortest chain along the pathway from the first atom of the linker (bonded to a 3' substituent of the preceding nucleotide, such as oxygen or phosphate) to the last atom of the linker (bonded to a 5' substituent of the following nucleotide, such as oxygen or phosphate) (e.g., from ~ to # in the structure of formula (I) below). The table below provides approximate predicted bridging lengths for various linkers.

[0347] Exemplary linker lengths predicted by atomic number, number of ethylene glycol units, approximate linker length (in Å) assuming an ethylene glycol monomer length of about 3.7 Å, and suitable positions for at least the entire ring portion of the substituted hairpin structure are provided in Table 8 below. Substitution of two nucleotides requires a linker length of at least about 11 Å. Substitution of at least three nucleotides requires a linker length of at least about 16 Å.

[0348] Table 9A

[0349] In some implementations, the internal connector includes the structure of form (I): ~-L0-L1-L2-# (I) in: ~ indicates a bond with the 3' substituent of the preceding nucleotide; # indicates a bond with the 5' substituent of the following nucleotide; L0 is empty or C 1-3 Aliphatic groups; L1 is -[E 1 -(R 1 )] m -,in Each R 1 C is independent 1-5 Aliphatic groups, optionally surrounded by 1 or 2 E 2 replace, Each E 1 and E 2 Independently, they are hydrogen bond acceptors, or each is independently selected from cyclic and heterocyclic hydrocarbons, and Each m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and L2 is empty, C 1-3 Aliphatic groups, or hydrogen bond acceptors.

[0350] In some embodiments, L1 comprises one or more -CH2CH2O-, -CH2OCH2-, or -OCH2CH2- units (“ethylene glycol subunits”). In some embodiments, the number of -CH2CH2O-, -CH2OCH2-, or -OCH2CH2- units is in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0351] In some implementations, m is 1, 2, 3, 4, or 5. In some implementations, m is 1, 2, or 3. In some implementations, m is 6, 7, 8, 9, or 10.

[0352] In some implementations, L0 is empty. In some implementations, L0 is -CH2- or -CH2CH2-.

[0353] In some implementations, L2 is empty. In some implementations, L2 is -O-, -S-, or C. 1-3 Aliphatic group. In some embodiments, L2 is -O-. In some embodiments, L2 is -S-. In some embodiments, L2 is -CH2- or -CH2CH2-.

[0354] In the table of this paper, L1 and L2 are arbitrarily C9 and C18, respectively, as follows: In some embodiments, the internal linker has a bridging length of about 3-30 atoms, optionally 12-21 atoms, and the linker replaces at least 2 nucleotides of the gRNA. In some embodiments, the internal linker has a bridging length of about 6-18 atoms, optionally about 6-12 atoms, and the linker replaces at least 2 nucleotides of the gRNA. In some embodiments, the internal linker replaces 2-12 nucleotides.

[0355] In some embodiments, the guide RNA comprises the nucleic acid sequence of SEQ ID NO: 700, including modifications disclosed elsewhere herein. Table 9B shows various embodiments of gRNA structures and types with possible numbers and positions of internal linkers.

[0356] Table 9B. In some embodiments, the internal linker is located in the repeat-inverse region of the gRNA. In some embodiments, the internal linker replaces at least four nucleotides in the repeat-inverse region of the gRNA. In some embodiments, the internal linker replaces the loop in the repeat-inverse region of the Nme Cas9 gRNA, corresponding to nucleotides 49-52 in SEQ ID NO: 700.

[0357] In some implementations, the internal linker replaces 2, 3, or 4 nucleotides of the linker region of the gRNA.

[0358] In some embodiments, the internal linker replaces the loop in the hairpin 1 region of the Nme Cas9 gRNA, corresponding to nucleotides 87-90 in SEQ ID NO: 700. In some embodiments, the internal linker replaces at least four nucleotides of the loop in the hairpin 2 region of the Nme Cas9 gRNA, corresponding to nucleotides 122-125 in SEQ ID NO: 700. In some embodiments, the internal linker replaces the loop in the hairpin 1 region of the Nme Cas9 gRNA (corresponding to nucleotides 87-90 in SEQ ID NO: 700) and at least four nucleotides of the loop in the hairpin 2 region of the Nme Cas9 gRNA (corresponding to nucleotides 122-125 in SEQ ID NO: 700).

[0359] Table 9C. Exemplary NmeCas9 guide RNAs containing linkers

[0360] Nucleotide modifications in the modified sequence are indicated in Table 9C as follows: where “m” indicates a 2'-O-Me modification, “*” indicates a phosphate thioester bond between nucleotides, and within each individually specified nucleotide, no modification indicates RNA with a phosphodiesterase backbone (2'-OH). Even in the case of modified sequences, each nucleotide of (N)20-25 is optionally modified independently. In some instances, at least the first three nucleotides are modified, for example, (mN*)3(N)17-22.

[0361] In some embodiments, a composition is provided comprising one or more guide RNAs containing a guide sequence of any of those in Table 2. In some embodiments, a composition is provided comprising one or more guide RNAs containing a guide sequence of any of those in Table 2, wherein the nucleotide of SEQ ID: 706 follows the guide sequence at its 3' end. In some embodiments, one or more guide RNAs containing a guide sequence of any of those in Table 2 (wherein the nucleotide of SEQ ID NO: 706 follows the guide sequence at its 3' end) are modified according to a modification pattern of any of those in SEQ ID NO: 710-715. In some embodiments, one or more guide RNAs containing a guide sequence of any of those in Table 2 (wherein the nucleotide of SEQ ID NO: 706 follows the guide sequence at its 3' end) are modified according to a modification pattern of any of the sequences shown in Table 6 (e.g., SEQ ID NO: 712 or 713). In some embodiments, one or more guide RNAs containing the guide sequence of any of those in Table 2 (where the nucleotide of SEQ ID NO: 706 follows the guide sequence at its 3' end) are modified according to the modification pattern of any of those in SEQ ID NO: 713.

[0362] In some embodiments, an sgRNA is provided comprising a guide sequence of any of those listed in Table 2 and any conserved portion of the sgRNAs shown in Tables 6-7, optionally having a modification pattern of any of the sgRNAs shown in Table 7, optionally wherein the sgRNA comprises 5' and 3' end modifications (if not already shown in the constructs in Table 7).

[0363] In some implementations, the sgRNA contains any of the modification patterns shown in Table 7 below, where N is any natural or non-natural nucleotide, and where the entire N constitutes the guide sequence as described in Table 2 herein. Table 7 does not depict the guide sequence portion of the sgRNA. Although N is replaced by a nucleotide of the guide sequence, the modification remains as shown in Table 7. That is, although the guide nucleotide replaces “N,” the nucleotide is modified as shown in Table 7.

[0364] In some embodiments, an sgRNA is provided comprising a guide sequence of any of those listed in Table 2 (e.g., SEQ ID NO: 1-49) and any conserved portions of the sgRNAs shown in Tables 4-7, optionally having a modification pattern of any of the sgRNAs shown in Tables 6 and 7, optionally wherein the sgRNA comprises 5' and 3' end modifications (if not already shown in the constructs in Table 7).

[0365] In some implementations, the sgRNA contains any of the modification patterns shown in Table 7 below, where N is any natural or non-natural nucleotide, and where the entire N constitutes the guide sequence as described in Table 2 herein. Table 7 does not depict the guide sequence portion of the sgRNA. Although N is replaced by a nucleotide of the guide sequence, the modification remains as shown in Table 7. That is, although the guide nucleotide replaces “N,” the nucleotide is modified as shown in Table 7.

[0366] In some embodiments, a composition is provided comprising one or more guide RNAs, the guide RNAs comprising a guide sequence of any of the nucleic acids in Table 2. In one aspect, a composition is provided comprising one or more gRNAs, the gRNAs comprising a guide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identity with any of the nucleic acids in Table 2.

[0367] In other embodiments, a composition is provided comprising at least one (e.g., at least two) gRNAs, the gRNAs comprising a guide sequence selected from any two or more of the guide sequences shown in Table 2. In some embodiments, the composition comprises at least two gRNAs, each of the gRNAs comprising a guide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identity with any of the guide sequences shown in Table 2.

[0368] In some embodiments, the guide RNA compositions of this disclosure are designed to recognize (e.g., hybridize to) a target sequence. For example, the target sequence can be recognized and cleaved by a Cas lysin containing the guide RNA. In some embodiments, an RNA-guided DNA binder (such as a Cas lysin) can be directed by the guide RNA to the target sequence, wherein the guide sequence of the guide RNA hybridizes with the target sequence and the RNA-guided DNA binder (such as a Cas lysin) cleaves the target sequence.

[0369] In some embodiments, the selection of one or more guide RNAs is determined based on a target sequence within the target gene. In some embodiments, compositions containing one or more guide sequences contain guide sequences complementary to the corresponding genomic regions shown in Table 2, based on coordinates from the human reference genome hg38. In other embodiments, the guide sequence may be complementary to a sequence within the target gene closely adjacent to the genomic coordinates listed in Table 2. For example, in other embodiments, the guide sequence may be complementary to a sequence containing 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2. Without being bound by any particular theory, modifications in certain regions of the target gene (e.g., frameshift mutations caused by insertions / deletions, occurring as a result of nuclease-mediated DSBs) may be less permissible than mutations in other regions; therefore, the location of the DSB is an important factor in the amount or type of protein knockdown that may result. In some embodiments, gRNAs complementary to or having complementary properties to the target sequence within the target gene are used to direct RNA-guided DNA binders to specific locations within the target gene.

[0370] In some embodiments, the guide sequence has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, or 80% identity with the target sequence present in the target gene. In some embodiments, the guide sequence has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, or 80% identity with the target sequence present in the human target gene.

[0371] In some embodiments, the target sequence may be complementary to the guide sequence of the guide RNA. In some embodiments, the complementarity or identity between the guide sequence of the guide RNA and its corresponding target sequence may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, or 4 mismatches, wherein the total length of the guide sequence is 20 nucleotides. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches, wherein the guide sequence is 20 nucleotides long.

[0372] VI. RNA-guided DNA binding agents In some embodiments, the compositions or formulations disclosed herein comprise mRNA containing an open reading frame (ORF) encoding an RNA-guided DNA binder, such as the Cas nuclease described herein. In some embodiments, mRNA comprising an ORF encoding an RNA-guided DNA binder (such as a Cas nuclease) is provided, used, or administered.

[0373] In some embodiments, the composition further comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0374] In some implementations, the nucleic acid encoding an RNA-guided DNA binder comprises mRNA containing an open reading frame (ORF) encoding an RNA-guided DNA binder.

[0375] In some implementations, the RNA-guided DNA binder is a nuclease.

[0376] In some implementations, the RNA-guided DNA binder is the Cas9 nuclease.

[0377] In some implementations, Cas9 is Nme Cas9.

[0378] In some embodiments, Nme Cas9 comprises an amino acid sequence having at least 90% identity with the sequence selected from SEQ ID NO: 832-834, or an ORF encoding Nme Cas9 having at least 90% identity with the sequence selected from SEQ ID NO: 832-834. In some embodiments, Nme Cas9 comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 832, or an ORF encoding Nme Cas9 having at least 90% identity with SEQ ID NO: 832.

[0379] In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity with the sequence selected from SEQ ID NO: 802-810. In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity with SEQ ID NO: 802.

[0380] In some implementations, the nuclease has double-stranded endonuclease activity.

[0381] In some implementations, the nuclease has nicking enzyme activity.

[0382] In some implementations, the nuclease is non-catalytically active.

[0383] In some implementations, nucleases also include heterologous functional domains.

[0384] In some implementations, the nuclease is a nicking enzyme and the heterologous functional domain is a deaminase.

[0385] In some implementations, the deaminase is cytidine deaminase or adenine deaminase.

[0386] In some implementations, the deaminase is cytidine deaminase.

[0387] In some implementations, the deaminase is an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.

[0388] In some embodiments, the nuclease and deaminase comprise an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 831, 835-838 or an ORF encoding an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 831, 835-838.

[0389] In some implementations, the ORF encoding the amino acid sequence has at least 85% identity with SEQ ID NO: 801 or 804.

[0390] In some embodiments, the compositions described herein further comprise a uracil glycosidase inhibitor (UGI) or a nucleic acid encoding a UGI, wherein the nuclease polypeptide does not contain a UGI, or the nucleic acid encoding the polypeptide does not encode a UGI.

[0391] In some embodiments, the UGI comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 859 or 860, or an ORF encoding an amino acid sequence having at least 90% identity with SEQ ID NO: 859 or 860.

[0392] In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity with the sequence selected from SEQ ID NO: 823-826, optionally SEQ ID NO: 823.

[0393] In some implementations, the ORF is a modified ORF.

[0394] The RNA-guided DNA binders described herein cover Neisseria meningitidis Cas9 (NmeCas9) and its modified forms and variants. In some embodiments, NmeCas9 is Nme2 Cas9. In some embodiments, NmeCas9 is Nme1 Cas9. In some embodiments, NmeCas9 is Nme3 Cas9.

[0395] Modifications having an inactive catalytic domain (RuvC or HNH) are called "nicking enzymes." Nicking enzymes cleave only one strand of the target DNA, resulting in a single-strand break. A single-strand break can also be referred to as a "nick." In some embodiments, the compositions and methods comprise a nicking enzyme. In some embodiments, the compositions and methods comprise a nicking enzyme RNA-guided DNA binder, such as nicking enzyme Cas, for example nicking enzyme Cas9, which induces a nick rather than a double-strand break in the target DNA.

[0396] In some implementations, the NmeCas9 nuclease can be modified to contain only one functional nuclease domain. For example, the RNA-guided DNA binder can be modified to mutate or completely or partially delete one of the nuclease domains, thereby reducing its nucleic acid cleavage activity.

[0397] In some embodiments, an NmeCas9 nickase with a reduced-activity RuvC domain is used. In some embodiments, an NmeCas9 nickase with an inactive RuvC domain is used. In some embodiments, an NmeCas9 nickase with a reduced-activity HNH domain is used. In some embodiments, an NmeCas9 nickase with an inactive HNH domain is used.

[0398] In some implementations, the nuclease is modified to induce point mutations or base changes, for example, via deamination.

[0399] In some embodiments, the Cas protein comprises a fusion protein containing a Cas nuclease (e.g., NmeCas9) linked to a heterologous functional domain, which is either a nicking enzyme or non-catalytically active. In some embodiments, the Cas protein comprises a fusion protein containing a non-catalytically active Cas nuclease (e.g., NmeCas9) linked to a heterologous functional domain (see, for example, WO2014152432). In some embodiments, the non-catalytically active Cas9 is derived from Neisseria meningitidis Cas9. In some embodiments, the non-catalytically active Cas contains a mutation that deactivates Cas.

[0400] In some implementations, the heterologous functional domain is a domain that modifies gene expression, histones, or DNA. In some implementations, the heterologous functional domain is a transcriptional activation domain or a transcriptional repression domain. In some implementations, the nuclease is a non-catalytically active Cas nuclease, such as dCas9.

[0401] In some embodiments, the heterologous functional domain is a deaminase, such as cytidine deaminase or adenine deaminase. In some embodiments, the heterologous functional domain is a C-to-T base transition enzyme (cytidine deaminase), such as apolipoprotein B mRNA editing enzyme (APOBEC) deaminase. The heterologous functional domain (such as a deaminase) may be part of a fusion protein with a Cas nuclease having nicking enzyme activity or a non-catalytically active Cas nuclease discussed further below.

[0402] The RNA-guided DNA binders disclosed herein may also include base editing domains, such as deaminase domains, which introduce specific modifications into target nucleic acids.

[0403] In some embodiments, a nucleic acid is provided that includes an open reading frame encoding a polypeptide, the polypeptide including a cytidine deaminase (e.g., A3A), a C-terminal NmeCas9 nickase, and a first nuclear localization signal (NLS), wherein the polypeptide does not contain a uracil glycosidase inhibitor (UGI).

[0404] In some embodiments, the second NLS is located at the N-terminus of the NmeCas9 nickase. In some embodiments, the deaminase is located at the N-terminus of the NLS (i.e., the first or second NLS). In some embodiments, the deaminase is located at the N-terminus of all NLS in the polypeptide. In some embodiments, the deaminase is located at the N-terminus of all NLS in the polypeptide, wherein the polypeptide does not contain a uracil glycosidase inhibitor (UGI).

[0405] In some embodiments, the polynucleotide is DNA or RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, a polypeptide encoded by mRNA is provided.

[0406] In some implementations, the peptide containing A3A and RNA-guided nickase does not contain a uracil glycosidase inhibitor (UGI).

[0407] In some embodiments, a composition is provided comprising a first polypeptide or mRNA encoding the first polypeptide, the first polypeptide comprising a cytidine deaminase, optionally APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and optionally a second NLS; wherein the first NLS and the second NLS (when present) are located at the N-terminus of the sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not contain a uracil glycosidase inhibitor (UGI); and a second polypeptide or mRNA encoding the second polypeptide, the second polypeptide comprising a uracil glycosidase inhibitor (UGI), wherein the second polypeptide is different from the first polypeptide.

[0408] In some embodiments, a method for modifying a target gene is provided, the method comprising administering the composition described herein. In some embodiments, the method comprises delivering to a cell a first nucleic acid comprising a first open reading frame encoding a first polypeptide, the first polypeptide comprising a cytidine deaminase, optionally APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and optionally a second NLS; wherein the first NLS and the second NLS (when present) are located at the N-terminus of a sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not contain a uracil glycosidase inhibitor (UGI), and a second nucleic acid comprising a second open reading frame encoding a uracil glycosidase inhibitor (UGI), wherein the second nucleic acid is different from the first nucleic acid.

[0409] In some embodiments, the method includes delivering a polypeptide or a nucleic acid encoding the polypeptide to a cell, the polypeptide comprising a deaminase, optionally APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and a second NLS; wherein the first NLS and the second NLS are located at the N-terminus of the sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not contain a uracil glycosidase inhibitor (UGI), and delivering a uracil glycosidase inhibitor (UGI) or a nucleic acid encoding the UGI to the cell.

[0410] In some embodiments, the molar ratio of the mRNA encoding UGI to the mRNA encoding APOBEC3A deaminase (A3A) and the RNA-guided nickase is about 1:35 to about 30:1. In some embodiments, the molar ratio of the mRNA encoding UGI to the mRNA encoding APOBEC3A deaminase (A3A) and the RNA-guided nickase is not about 1:1.

[0411] Similarly, in some implementations, if the protein is delivered, the molar ratio of the mRNA encoding the UGI protein discussed above to the mRNA encoding the APOBEC3A deaminase (A3A) and the RNA-guided nickase is similar.

[0412] In some embodiments, the compositions described herein further comprise at least one gRNA. In some embodiments, the compositions described herein further comprise two gRNAs. In some embodiments, a composition is provided comprising the mRNA described herein and at least one gRNA (e.g., two gRNAs). In some embodiments, the gRNA is a single-guide RNA (sgRNA). In some embodiments, the gRNA is a dual-guide RNA (dgRNA).

[0413] In some embodiments, the composition enables genome editing after administration to a subject.

[0414] Cytidine deaminase; APOBEC3A deaminase Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and particularly enzymes of the APOBEC family (enzymes of the APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups), activation-induced cytidine deaminases (AID or AICDA), and CMP deaminases (see, for example, Conticello et al., Mol. Biol. Evol. 22:367-77, 2005; Conticello, Genome Biol. 9:229, 2008; Muramatsu et al., J. Biol. Chem. 274:18470-6, 1999; and Carrington et al., Cells 9:1690 (2020)).

[0415] In some embodiments, the cytidine deaminases disclosed herein are enzymes of the APOBEC family. In some embodiments, the cytidine deaminases disclosed herein are enzymes of the APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups. In some embodiments, the cytidine deaminases disclosed herein are enzymes of the APOBEC3 subgroup. In some embodiments, the cytidine deaminases disclosed herein are APOBEC3A deaminases (A3A). In some embodiments, the deaminase comprises APOBEC3A deaminase.

[0416] In some embodiments, the APOBEC3A deaminase (A3A) disclosed herein is human A3A. In some embodiments, the A3A is wild-type A3A.

[0417] In some embodiments, A3A is an A3A variant. The A3A variant shares homology with wild-type A3A or fragments thereof. In some embodiments, the A3A variant has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity with wild-type A3A. In some implementations, the A3A variants may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid variations compared to the wild-type A3A. In some implementations, the A3A variant comprises a fragment of A3A such that the fragment has at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with the corresponding fragment of wild-type A3A.

[0418] In some embodiments, the A3A variant is a protein having a sequence that differs from the wild-type A3A protein due to one or more mutations (such as substitution, deletion, insertion, or one or more single-point substitutions). In some embodiments, a shortened A3A sequence may be used, for example, by deleting the N-terminus, C-terminus, or internal amino acids. In some embodiments, a shortened A3A sequence is used, wherein one to four amino acids are deleted at the C-terminus of the sequence. In some embodiments, APOBEC3A (such as human APOBEC3A) has wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, APOBEC3A (such as human APOBEC3A) has asparagine at amino acid position 57 (as numbered in the wild-type sequence).

[0419] In some implementations, wild-type A3A is human A3A (UniPROT accession ID: p31941, SEQ ID NO: 850).

[0420] In some embodiments, the A3A disclosed herein comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 850. In some embodiments, the identity level is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, A3A comprises an amino acid sequence having at least 87% identity with SEQ ID NO: 850. In some embodiments, A3A comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 850. In some embodiments, A3A comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 850. In some embodiments, A3A comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 850. In some embodiments, A3A comprises an amino acid sequence having at least 99% identity with A3A ID NO: 850. In some embodiments, A3A comprises the amino acid sequence of SEQ ID NO: 850.

[0421] In some embodiments, the cytidine deaminase disclosed herein comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 850.

[0422] In some embodiments, any of the aforementioned levels of identity is at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the UGI comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 859 or 860. In some embodiments, the UGI comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 859 or 860. In some embodiments, the UGI comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 859 or 860. In some embodiments, the UGI comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 859 or 860. In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NO: 859 or 860.

[0423] connector In some embodiments, the polypeptide comprising a deaminase and an RNA-guided nickase described herein further comprises a linker connecting the deaminase and the RNA-guided nickase. In some embodiments, the linker is a peptide linker. In some embodiments, the nucleic acid encoding the polypeptide comprising the deaminase and the RNA-guided nickase further comprises a sequence encoding the peptide linker. In some embodiments, mRNA encoding a deaminase-linker-RNA-guided nickase fusion protein is provided.

[0424] In some embodiments, the peptide linker is any amino acid segment having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 or more amino acids.

[0425] In some embodiments, the peptide linker is a 16-residue “XTEN” linker or a variant thereof (see, for example, examples; and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequences SGSETPGTSESATPES (SEQ ID NO: 901), SGSETPGTSESA (SEQ ID NO: 902), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 903).

[0426] In some embodiments, the peptide linker comprises (GGGGS)n (SEQ ID NO: 931), (G)n, (EAAAK)n (SEQ ID NO: 932), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 901) motif (see, for example, Guilinger JP, Thompson DB, Liu D R. Fusion of catalytically inactive Cas9to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) or (XP)n motif, or a combination of any of these sequences, wherein n is independently an integer between 1 and 30. See WO2015089406, for example, paragraph

[0012] , the entire contents of which are incorporated herein by reference.

[0427] In some embodiments, the peptide linker comprises one or more sequences selected from SEQ ID NO: 901-991.

[0428] VII. Modified gRNAs and mRNAs In some embodiments, the gRNA is chemically modified. A gRNA containing one or more modified nucleosides or nucleotides is referred to as a "modified" gRNA or "chemically modified" gRNA to describe the presence of one or more non-natural or naturally occurring components or conformations used in place of or with the addition of canonical A, G, C, and U residues. In some embodiments, the modified gRNA is synthesized using non-canonical nucleosides or nucleotides (referred to herein as "modified"). Modified nucleosides and nucleotides may include one or more of the following: (i) altering (e.g., replacing) one or two non-linked phosphate oxygens or one or more linked phosphate oxygens in the phosphodiester backbone (exemplary backbone modification); (ii) altering (e.g., replacing) a component of the ribose (e.g., the 2' hydroxyl group on the ribose) (exemplary sugar modification); (iii) modifying or replacing a naturally occurring nucleobase, including using a non-canonical nucleobase (exemplary base modification); and (iv) modifying the 3' or 5' end of an oligonucleotide to provide exonuclease stability, for example, by modifying the ribose with a 2' O-me, 2' halide, or 2' deoxy-substituted ribose; or a reverse base-free nucleotide, or by replacing the phosphodiester with a thiophosphate.

[0429] Chemical modifications (such as those listed above) can be combined to provide modified gRNA or mRNA containing two, three, four, or more modified nucleosides and nucleotides (collectively, “residues”). For example, the modified residues may have modified sugars and modified nucleobases. In some embodiments, up to 15% of the phosphate groups of the gRNA molecule are replaced by thiophosphate groups. In some embodiments, the modified gRNA contains at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA contains at least one modified residue at or near the 3' end of the RNA.

[0430] In some embodiments, the gRNA contains one, two, three, or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, 10%, 15%, preferably at least 20%, 25%, 30%, 35%, 40%, 45%, or 50%) of the modified gRNA is a modified nucleoside or nucleotide. In some embodiments, at least 5% of the modified guide RNA is a modified nucleotide or nucleoside. In some embodiments, at least 10% of the modified guide RNA is a modified nucleotide or nucleoside. In some embodiments, at least 15% of the modified gRNA is a modified nucleotide or nucleoside. In some embodiments, preferably at least 20% of the modified gRNA is a modified nucleotide or nucleoside. In some embodiments, no more than 65% of the modified gRNA is a modified nucleotide. In some embodiments, no more than 55% of the modified gRNA is a modified nucleotide. In some embodiments, no more than 50% of the modified gRNA is a modified nucleotide. In some embodiments, 10-70% of the modified gRNA is a modified nucleotide. In some embodiments, 20-70% of the modified gRNA consists of modified nucleotides. In some embodiments, 20-50% of the modified gRNA consists of modified nucleotides, and the nuclease is an Nme Cas9 nuclease. In some embodiments, 30-70% of the modified gRNA consists of modified nucleotides, and the nuclease is an Nme Cas9 nuclease.

[0431] Unmodified nucleic acids may be readily degraded by, for example, intracellular nucleases or those found in serum. For instance, nucleases can hydrolyze the phosphodiester bonds of nucleic acids. Therefore, in one aspect, the gRNAs described herein may contain one or more modified nucleosides or nucleotides, for example, to introduce stability against intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein may exhibit a reduced innate immune response when introduced into cell populations in vivo and in vitro. The term "innate immune response" includes cellular responses to exogenous nucleic acids (including single-stranded nucleic acids) involving the induction of cytokine expression and release (especially interferon) and cell death.

[0432] In some embodiments of main-chain modification, the phosphate ester groups of the modified residues can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, the modified residues (e.g., modified residues present in modified nucleic acids) can include replacing unmodified phosphate ester moieties with modified phosphate ester groups as described herein. In some embodiments, main-chain modification of the phosphate ester backbone can include alterations that produce uncharged linkers or charged linkers with asymmetric charge distributions.

[0433] Examples of modified phosphate groups include thiophosphates, boron phosphates, methylphosphonates, aminophosphates, dithiophosphates, alkyl or aryl phosphonates, and phosphate triesters. The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the aforementioned atoms or groups can make the phosphorus atom chiral. The stereoisomer source phosphorus atom can have an "R" configuration (here, Rp) or an "S" configuration (here, Sp). The backbone can also be modified by replacing bridging oxygens (i.e., linking the phosphate ester to the oxygen of the nucleoside) with nitrogen (bridging aminophosphates), sulfur (bridging thiophosphates), and carbon (bridging methylenephosphonates). Substitution can occur at any of the linked oxygens or at both linked oxygens.

[0434] In certain main-chain modifications, the phosphate ester group may be replaced by a phosphorus-free linking group (e.g., an amide bond). In some embodiments, the charged phosphate ester group may be replaced by a neutral portion. Examples of portions that can replace the phosphate ester group include, but are not limited to, methylphosphonates, carboxymethyl groups, carbamates, amides, and thioethers. Other examples of portions that can replace the phosphate ester group include, but are not limited to, ethylene oxide linkers, sulfonates, sulfonamides, thiomethyl acetals, methyl acetals, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino.

[0435] Nucleic acid-mimicking scaffolds can also be constructed, where phosphate linkers and ribose are replaced by nuclease-resistant nucleosides or nucleotide substitutes. Such modifications can include backbone and sugar modifications. In some embodiments, nucleobases can be tethered by alternative backbones. Examples may include, but are not limited to, morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside substitutes.

[0436] Modified nucleosides and modified nucleotides can include one or more modifications to the glycosyl group, i.e., sugar modifications. For example, the 2' hydroxyl group (OH) can be modified, for instance, by being replaced with many different "oxygen" or "deoxy" substituents. In some embodiments, modification of the 2' hydroxyl group can enhance the stability of the nucleic acid because the hydroxyl group can no longer be deprotonated to form a 2'-alkoxide ion.

[0437] Examples of 2' hydroxyl modification may include alkoxy or aryloxy (OR, where "R" can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CH2O). n CH2CH2OR, where R can be, for example, H or an optionally substituted alkyl group, and n can be an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20). In some embodiments, the 2' hydroxyl modification can be 2'-O-Me. In some embodiments, the 2' hydroxyl modification can be a 2'-fluorine modification, wherein the modification replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl modification can include a "locked" nucleic acid (LNA), wherein the 2' hydroxyl group can be, for example, a C1-6 alkylene group or a C1-6 alkylene group. 1-6 A heteroalkyl bridge is attached to the 4' carbon of the same ribose, wherein exemplary bridges may include methylene, propylene, ether, or amino bridges; O-amino (wherein the amino group may be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2). n-Amino group (wherein the amino group can be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl modification can include an "unlocked" nucleic acid (UNA) where the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl modification can include methoxyethyl (MOE), (OCH2CH2OCH3, for example, a PEG derivative). The 2' modification can include hydrogen (i.e., deoxyribose); a halogen (e.g., bromine, chlorine, fluorine, or iodine); an amino group (wherein the amino group can be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH (CH2CH2NH). n CH2CH2-amino (wherein the amino group may be, for example, as described herein), -NHC(O)R (wherein R may be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups, which may optionally be substituted with amino groups, for example, as described herein.

[0438] Sugar modifications can include a glycosyl group, which may also contain one or more carbon atoms having a stereochemical configuration opposite to that of the corresponding carbon atom in ribose. Therefore, modified nucleic acids can include nucleotides containing, for example, arabinose as a sugar. Modified nucleic acids can also include abasic sugars. These abasic sugars can be further modified at one or more constitutive sugar atoms. Modified nucleic acids can also include one or more sugars in the L-form, such as L-nucleosides. As used herein, a single abasic sugar should not be construed as causing a break in the double strand.

[0439] In some embodiments, the 2' modification includes, for example, modifications including 2'-OMe, 2'-F, 2'-H, and optionally 2'-O-Me.

[0440] The modified nucleosides and modified nucleotides described herein that can be incorporated into the modified nucleic acid may include modified bases, also known as nucleobases. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases may be modified or entirely substituted to provide modified residues that can be incorporated into the modified nucleic acid. The nucleobases of the nucleotide may be independently selected from purines, pyrimidines, purine analogs, and pyrimidine analogs. In some embodiments, the nucleobases may include, for example, naturally occurring and synthetic derivatives of the base.

[0441] In embodiments employing dual guide RNA, each of the crRNA and tracrRNA may contain modifications. Such modifications may be at one or both ends of the crRNA or tracrRNA. In embodiments containing sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, or internal nucleotides may be modified, or the entire sgRNA may be chemically modified. Some embodiments include a 5' end modification. Some embodiments include a 3' end modification. Some embodiments include both 5' and 3' end modifications.

[0442] In some embodiments, the guide RNA disclosed herein comprises one of the structural / modification patterns disclosed in US20170114334, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNA disclosed herein comprises one of the structural / modification patterns disclosed in WO2017 / 136794, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNA disclosed herein comprises one of the modification patterns disclosed in WO2018 / 107028, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNA disclosed herein comprises one of the structural / modification patterns disclosed in WO2023081687A1, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNA disclosed herein comprises one of the structural / modification patterns disclosed in WO2022261292, the contents of which are hereby incorporated by reference in their entirety.

[0443] The terms “mA”, “mC”, “mU”, or “mG” can be used to denote nucleotides modified with 2'-O-Me. The terms “fA”, “fC”, “fU”, or “fG” can be used to denote nucleotides substituted with 2'-F. “*” can be used to depict PS modifications.

[0444] The terms A*, C*, U*, or G* can be used to denote a nucleotide that is linked to the next (e.g., 3') nucleotide via a PS bond.

[0445] The terms “mA*”, “mC*”, “mU*”, or “mG*” can be used to denote a nucleotide that has been replaced by 2'-O-Me and linked to the next (e.g., 3') nucleotide via a PS bond.

[0446] Any of the modifications described below may be present in the gRNA and mRNA described herein.

[0447] In the case of chemically modified sequences, “A”, “C”, “G”, “N” and “U” represent RNA nucleotides, that is, nucleotides with a 2’-OH group that is linked to a phosphodiesterase bond with a 3’ nucleotide.

[0448] The terms “mA”, “mC”, “mU”, or “mG” are used to denote adenine, cytosine, uridine, or guanidine nucleotides that have been modified with 2'-O-Me, respectively.

[0449] The modification of 2'-O-methyl can be described as follows: Another chemical modification that has been shown to affect the sugar ring of nucleotides is halogen substitution. For example, 2'-fluorine (2'-F) substitution on the sugar ring of nucleotides can increase oligonucleotide binding affinity and nuclease stability.

[0450] In this application, the terms “fA”, “fC”, “fU”, or “fG” are used to denote nucleotides that have been substituted with 2'-F.

[0451] The substitution of 2'-F can be described as follows: A phosphate thioester (PS) bond or linkage refers to a non-bridging phosphate oxygen bond in a phosphate diester linkage, such as a bond between nucleotide bases. When phosphate thioesters are used to produce oligonucleotides, the modified oligonucleotides can also be called S-oligonucleotides.

[0452] The asterisk (*) is used to indicate a PS modification. In this application, the terms A*, C*, U*, or G* may be used to indicate a nucleotide linked to the next (e.g., 3') nucleotide via a PS bond.

[0453] In this application, the terms “mA*”, “mC*”, “mU*” or “mG*” are used to denote a nucleotide that has been replaced with 2'-O-Me and linked to the next (e.g., 3') nucleotide via a PS bond.

[0454] The diagram below illustrates how S-substitution into a non-bridging phosphate group produces a PS bond that replaces the phosphodiester bond: Abase-free nucleotides are nucleotides that lack a nitrogenous base. The image below depicts an oligonucleotide whose abase-free (also known as apurinol) site is missing a base. As used herein, the presence of a single abase-free site should not be considered a disruption of the duplex, such as the duplex formed between the guide sequence of a guide RNA and its target site in the genome: A reverse base is a base that has a bond that is the reverse of the normal 5' to 3' bond (i.e., a 5' to 5' bond or a 3' to 3' bond). Such reverse bases exist only as terminal nucleotides. In 3' to 5' chemical synthesis methods, the reverse base does not have a 5' hydroxyl group that can be used to grow the chain. For example: Abase-free nucleotides can be linked by reverse bonding. For example, an abase-free nucleotide can be linked to a terminal 5' nucleotide via a 5'-5' bond, or a base-free nucleotide can be linked to a terminal 3' nucleotide via a 3'-3' bond. The reverse abase-free nucleotide at the terminal 5' or 3' nucleotide can also be called a reverse abase-free cap.

[0455] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' end and one or more of the last three, four, or five nucleotides at the 3' end are modified. In some embodiments, the modification is 2'-O-Me, 2'-F, a reverse abasic nucleotide, a PS bond, or other nucleotide modifications known in the art to increase stability or performance.

[0456] In some implementations, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked via phosphate thioester (PS) bonds.

[0457] In some embodiments, the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise nucleotides modified with 2'-O-methyl (2'-O-Me). In some embodiments, the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise nucleotides modified with 2'-fluorine (2'-F). In some embodiments, the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise inverted abase-free nucleotides.

[0458] In some embodiments, the Nme guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification patterns shown in Tables 7A-7B, for example... mN*mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNNmGUUGmUmAmGmCUCCCmUmGmAmAmCmCGUUmGmCUAmCAAU*AAGmGmCCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU (SEQ ID NO: 731); or mN*mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNNmGUUGmUmAmGmCUCCCmUmGmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU (SEQ ID NO: 732); Each of A, C, G, U, and N represents an RNA nucleotide, a 2'-OH group, and a phosphodiester bond to a 3' nucleotide; m indicates a 2'-O-methyl (2'-O-Me) modified nucleotide; and * indicates a phosphate thioester bond between nucleotides. The entirety of N constitutes a guide sequence that directs the nuclease to a target sequence in the target gene. In some embodiments, the guide sequence comprises the guide sequences shown in Tables 2-3.

[0459] As mentioned above, in some embodiments, the compositions or formulations disclosed herein comprise mRNA containing an open reading frame (ORF) encoding an RNA-guided DNA binder, such as a Cas nuclease, for example, the Cas9 nuclease described herein. In some embodiments, mRNA comprising an ORF encoding an RNA-guided DNA binder (such as a Cas nuclease, e.g., the Cas9 nuclease) is provided, used, or administered. In some embodiments, the ORF encoding an RNA-guided DNA nuclease is referred to as a “modified RNA-guided DNA binder ORF” or simply “modified ORF,” which is used as an abbreviation to indicate ORF modification.

[0460] In some embodiments, the mRNA or modified ORF may contain modified uridine at at least one, multiple, or all uridine positions. In some embodiments, the modified uridine is uridine modified at the 5-position, for example, with halogen, methyl, or ethyl. In some embodiments, the modified uridine is pseudouridine modified at the 1-position, for example, with halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or combinations thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methylpseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

[0461] In some embodiments, the mRNA disclosed herein includes a 5' cap, such as Cap0, Cap1, or Cap2. The 5' cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below for example with regard to ARCA), which is linked via a 5'-triphosphate to the 5' position of the first nucleotide of the 5' to 3' strand of the mRNA, i.e., the first cap proximal nucleotide. In Cap0, the ribose of both the first and second cap proximal nucleotides of the mRNA contains a 2'-hydroxyl group. In Cap1, the ribose of the first and second transcription nucleotides of the mRNA contains a 2'-methoxy group and a 2'-hydroxyl group, respectively. In Cap2, the ribose of both the first and second cap proximal nucleotides of the mRNA contains a 2'-methoxy group. See, for example, Katibah et al. (2014). Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA114(11):E2106-E2115. Most endogenous higher eukaryotic mRNAs (including mammalian mRNAs, such as human mRNAs) contain Cap1 or Cap2. Cap0 and other cap structures different from Cap1 and Cap2 may be immunogenic in mammals (such as humans) because components of the innate immune system (such as IFIT-1 and IFIT-5) recognize them as “non-self,” potentially leading to elevated levels of cytokines, including type I interferon. Components of the innate immune system (such as IFIT-1 and IFIT-5) may also competitively bind to mRNAs with caps other than Cap1 or Cap2 with eIF4E, potentially inhibiting mRNA translation.

[0462] Caps can be co-transcribed. For example, ARCA (Anti-reverse cap analog; Thermo Fisher Scientific catalog number AM8045) is a cap analog containing 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of a guanine ribonucleotide, which can be incorporated into the transcript at the in vitro initiation site. ARCA produces a Cap0 cap, where the 2' position of the first cap proximal nucleotide is a hydroxyl group. See, for example, Stepinski et al., (2001) "Synthesis and properties of mRNAs containing the novel 'anti-reverse' capanalogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG", RNA 7: 1486-1495. The ARCA structure is shown below.

[0463] CleanCap™ AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies catalog number N-7113) or CleanCap™ GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies catalog number N-7133) can be used to co-transcribe the Cap1 structure. The 3'-O-methylated forms of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies catalog numbers N-7413 and N-7433, respectively, or CleanCapAU is available from TriLink Biotechnologies catalog number N-7114. The CleanCap™ AG structure is shown below.

[0464] Alternatively, the cap can be added to the RNA post-transcriptionally. For example, vaccinia capping enzyme is commercially available (New England Biolabs catalog number M2080S) and possesses RNA triphosphatase and guanylate transferase activities provided by its D1 subunit and guanine methyltransferase activities provided by its D12 subunit. Therefore, it can add 7-methylguanine to RNA in the presence of S-adenosylmethionine and GTP to obtain Cap0. See, for example, Guo, P. and Moss, B. (1990). Proc. Natl. Acad. Sci . USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem 269, 24472-24479.

[0465] In some embodiments, the mRNA further comprises a polyadenylated (polyA) tail. In some embodiments, the polyA tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenine nucleotides, optionally up to 300 adenine nucleotides. In some embodiments, the polyA tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some embodiments, the polyA tail comprises non-adenine nucleotides, i.e., it is a discontinuous polyA tail. In some embodiments, the polyA tail is discontinuous by non-adenine nucleotides at approximately every 40, 50, 60, 70, 80, or 90 nucleotides. In some embodiments, the polyA tail is discontinuous by non-adenine nucleotides at approximately every 50 nucleotides.

[0466] VIII. Ribonucleoprotein complex In some embodiments, a composition is included comprising one or more sgRNAs, said sgRNAs comprising one or more guide sequences from Table 2 or one or more sgRNAs from Table 3, and an RNA-guided DNA binder, such as a nuclease, like a Cas nuclease, such as Cas9. In some embodiments, the RNA-guided DNA binder has lysinic activity, also referred to as double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binder comprises a Cas nuclease. Examples of Cas9 nucleases include those nucleases of the type II CRISPR system from Streptococcus pyogenes, Neisseria meningitidis, and other prokaryotes known in the art, and their modified (e.g., engineered or mutant) forms.

[0467] In some implementations, the Cas nuclease is the Cas9 nuclease derived from Neisseria meningitidis.

[0468] In some embodiments, the gRNA, together with the RNA-guided DNA binder, is referred to as a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA binder is a Cas nuclease. In some embodiments, the gRNA, together with the Cas nuclease, is referred to as a Cas RNP. In some embodiments, the RNP contains type I, type II, or type III components. In some embodiments, the Cas nuclease is the Cas9 protein from the type II CRISPR / Cas system. In some embodiments, the gRNA, together with Cas9, is referred to as a Cas9 RNP.

[0469] Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target DNA strand. In some embodiments, the Cas9 protein contains more than one RuvC domain or more than one HNH domain. In some embodiments, the Cas9 protein is wild-type Cas9. In each of the compositions, uses, and methods embodiments, Cas induces double-strand breaks in the target DNA.

[0470] In some embodiments, a chimeric Cas nuclease is used, wherein a domain or region of a protein is replaced by a portion of a different protein. In some embodiments, the Cas nuclease domain may be replaced by a domain from a different nuclease (such as Fok1). In some embodiments, the Cas nuclease may be a modified nuclease.

[0471] In other embodiments, the Cas nuclease may be derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of a cascade complex of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be the Cas3 protein. In some embodiments, the Cas nuclease may be derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0472] In some embodiments, the RNA-guided DNA binder has single-strand nicking enzyme activity, i.e., it can cleave one strand of DNA to produce a single-strand break, also known as a "nick". In some embodiments, the RNA-guided DNA binder comprises a Cas nicking enzyme. A nicking enzyme is an enzyme that creates a nick in dsDNA, i.e., it cuts one strand of the DNA double helix but not the other. In some embodiments, the Cas nicking enzyme is of the type of Cas nuclease (e.g., the Cas nuclease discussed above) in which the endonuclease active site is inactivated, for example by alteration of one or more catalytic domains (e.g., point mutation). See, for example, U.S. Patent No. 8,889,356, for example, the discussion of Cas nicking enzymes and exemplary catalytic domain alterations. In some embodiments, the Cas nicking enzyme (such as the Cas9 nicking enzyme) has an inactive RuvC or HNH domain.

[0473] In some embodiments, the RNA-guided DNA binder is modified to contain only one functional nuclease domain. For example, a modifiable protein can be used to mutate or completely or partially delete one of the nuclease domains to reduce its nucleic acid cleavage activity. In some embodiments, a nickase with a reduced-activity RuvC domain is used. In some embodiments, a nickase with an inactive RuvC domain is used. In some embodiments, a nickase with a reduced-activity HNH domain is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0474] In some embodiments, conserved amino acids within the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may contain amino acid substitutions within the RuvC or RuvC-like nuclease domain.

[0475] In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, an optional NLS, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase having amino acid substitutions in the HNH or HNH-like nuclease domain, such as the D16A NmeCas9 nickase. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, an optional NLS, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase having amino acid substitutions in the HNH or HNH-like nuclease domain, such as the D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a first NLS and a second NLS, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase having amino acid substitutions in the HNH or HNH-like nuclease domain, such as the D16A NmeCas9 nickase. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a first NLS and a second NLS, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase having amino acid substitutions in the HNH or HNH-like nuclease domain, such as the D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a first NLS, a cytidine deaminase (e.g., APOBEC3A), a second NLS, an optional linker, and an Nme Cas9 nickase having amino acid substitutions in the HNH or HNH-like nuclease domain, such as the D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a first NLS, a cytidine deaminase (e.g., APOBEC3A), a second NLS, an optional linker, and an Nme Cas9 nickase having amino acid substitutions in the HNH or HNH-like nuclease domain, such as the D16A Nme2Cas9 nickase.

[0476] In some embodiments, the mRNA encoding the nicking enzyme is provided in combination with a pair of guide RNAs complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nicking enzyme to the target sequence and introduce the DSB by creating a nick on the opposite strand of the target sequence (i.e., a double nick). In some embodiments, using a double nick can improve specificity and reduce off-target effects. In some embodiments, the nicking enzyme is used with two separate guide RNAs targeting opposite DNA strands to create a double nick in the target DNA. In some embodiments, the nicking enzyme is used with two separate guide RNAs selected to be very close to each other to create a double nick in the target DNA.

[0477] In some embodiments, the RNA-guided DNA binder lacks lyase and nicking enzyme activity. In some embodiments, the RNA-guided DNA binder comprises a dCas DNA-binding polypeptide. The dCas polypeptide has DNA-binding activity but is substantially lacking in catalytic (lyase / nicking enzyme) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA binder or dCas DNA-binding polypeptide lacking lyase and nicking enzyme activity is a type of Cas nuclease (e.g., the Cas nucleases discussed above) in which its endonuclease active site is inactivated, for example by one or more alterations (e.g., point mutations) in its catalytic domain. See, for example, US 20140186958; US 20150166980; and US 20190338308.

[0478] In some implementations, the RNA-guided DNA binder contains one or more heterologous functional domains (e.g., is or contains a fusion polypeptide).

[0479] In some embodiments, the heterologous functional domain facilitates the transport of the RNA-guided DNA binder to the cell nucleus. For example, the heterologous functional domain can be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA binder can be fused with 1-5 NLSs. In some embodiments, the RNA-guided DNA binder can be fused with 2, 3, or 4 NLSs. In some embodiments, the RNA-guided DNA binder can be fused with two NLSs. In some embodiments, the RNA-guided DNA binder can be fused with one NLS. When using one NLS, the NLS can be attached to the N-terminus or C-terminus of the RNA-guided DNA binder sequence. In some embodiments, the NLS is not attached to the C-terminus. The NLS can also be inserted within the RNA-guided DNA binder sequence. In some cases, at least two NLSs are identical (e.g., two SV40 NLSs). In some embodiments, the RNA-guided DNA binder contains at least two different NLSs. In some embodiments, the RNA-guided DNA binder is fused with two SV40 NLS sequences attached to the carboxyl terminus. In some embodiments, the RNA-guided DNA binder may fuse with two NLSs, one attached to the N-terminus and the other to the C-terminus. In some embodiments, the RNA-guided DNA binder may fuse with three NLSs. In some embodiments, the RNA-guided DNA binder may not fuse with any NLSs.

[0480] In some embodiments, the NLS may be an SV40 NLS. Exemplary SV40 NLS sequences may be SV40 NLS, PKKKRKV (SEQ ID NO: 916), or PKKKRRV (SEQ ID NO: 928). In some embodiments, the NLS may be a dichotomous sequence, such as the NLS of a nucleoplasmic protein, KRPAATKKAGQAKKKK (SEQ ID NO: 929). In some implementations, the NLS sequence may include LAAKRSRTT (SEQ ID NO: 917), QAAKRSRTT (SEQ ID NO: 918), PAPAKRERTT (SEQ ID NO: 919), QAAKRPRTT (SEQ ID NO: 920), RAAKRPRTT (SEQ ID NO: 921), AAAKRSWSMAA (SEQ ID NO: 922), AAAKRVWSMAF (SEQ ID NO: 923), AAAKRSWSMAF (SEQ ID NO: 924), AAAKRKYFAA (SEQ ID NO: 925), RAAKRKAFAA (SEQ ID NO: 926), or RAAKRKYFAV (SEQ ID NO: 927). The NLS may be a snurportin-1 infeedin-β (IBB domain), such as the SPN1-impβ sequence. See Huber et al., 2002, J. Cell Bio., 156, 467-479. In one specific embodiment, a single PKKKRKV (SEQ ID NO: 916). In some embodiments, the first NLS and the second NLS are independently selected from SV40 NLS, nucleoplasmic protein NLS, bipartite NLS, c-myc-like NLS, and NLS containing the sequence KTRAD (SEQ ID NO: 1004). In some embodiments, the first NLS and the second NLS may be the same (e.g., two SV40 NLS). In some embodiments, the first NLS and the second NLS may be different.

[0481] In some implementations, the first NLS is an SV40 NLS and the second NLS is a nucleoplasmic protein NLS.

[0482] In some embodiments, the SV40 NLS contains the sequence of PKKKRKVE (SEQ ID NO: 1005) or KKKRKVE (SEQ ID NO: 1006). In some embodiments, the nucleoplasmic protein NLS contains the sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 929). In some embodiments, the bipartite NLS contains the sequence of KRTADGSEFESPKKKRKVE (SEQ ID NO: 1007). In some embodiments, the c-myc-like NLS contains the sequence of PAAKKKKLD (SEQ ID NO: 1008).

[0483] One or more linkers may optionally be included at the fusion site of the NLS and the nuclease or between the NLS (when more than one NLS is present).

[0484] In some embodiments, one or more NLSs according to any of the foregoing embodiments are combined with one or more additional heterologous functional domains in the RNA-guided DNA binder. One or more linkers are optionally included at the fusion site.

[0485] In some embodiments, the heterofunctional domain may be able to alter the intracellular half-life of the RNA-guided DNA binder. In some embodiments, the half-life of the RNA-guided DNA binder may be increased. In some embodiments, the half-life of the RNA-guided DNA binder may be decreased. In some embodiments, the heterofunctional domain may be able to improve the stability of the RNA-guided DNA binder. In some embodiments, the heterofunctional domain may be able to decrease the stability of the RNA-guided DNA binder. In some embodiments, the heterofunctional domain may act as a signal peptide for protein degradation. In some embodiments, protein degradation may be mediated by proteases (such as proteasomes, lysosomal proteases, or calpases). In some embodiments, the heterofunctional domain may contain a PEST sequence. In some embodiments, the RNA-guided DNA binder may be modified by adding ubiquitin or polyubiquitin chains. In some embodiments, ubiquitin may be ubiquitin-like proteins (UBLs). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-associated modifier-1 (URM1), and developmentally downregulated protein-8 (NEDD8, expressed in neuronal precursor cells in Saccharomyces cerevisiae). S. cerevisiaeAlso known as Rub1), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and autophagy-12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane anchored UBL (MUB), ubiquitin fold modifier-1 (UFM1) and ubiquitin-like protein-5 (UBL5).

[0486] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope markers, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Suitable non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), and red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed). monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred) and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag or an epitope tag. Non-limiting exemplary tags include glutathione S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, biotinylate carboxyl carrier protein (BCCP), polyHis, and calmodulin. Non-limiting exemplary reporter genes include glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, or fluorescent protein.

[0487] In other embodiments, the heterologous functional domain may be an effector domain. When an RNA-guided DNA binder is directed to its target sequence, such as when a Cas nuclease is directed to its target sequence by gRNA, the effector domain may modify or influence the target sequence. In some embodiments, the effector domain may be selected from nucleic acid-binding domains, nuclease domains (e.g., non-Cas nuclease domains), epigenetic modification domains, transcriptional activation domains, and transcriptional repression domains. In some embodiments, the heterologous functional domain is a nuclease, such as FokI nuclease. See, for example, U.S. Patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, for example, Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression”. Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided geneactivation by CRISPR-Cas9-based transcription factors”, Nat. Methods 10:973-6(2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering”, Nat. Biotechnol. 31:833-8 (2013); Gilbert et al., “CRISPR-mediated modular RNA-guidedregulation of transcription in eukaryotes”, Cell 154:442-51 (2013). Therefore, RNA-guided DNA binders are essentially transformed into transcription factors, which can be guided by guide RNA to bind to desired target sequences.

[0488] In some embodiments, the heterologous functional domain is a deaminase, such as cytidine deaminase or adenine deaminase. In some embodiments, the heterologous functional domain is a C-to-T base transition enzyme (cytidine deaminase), such as apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.

[0489] In some embodiments, the heterologous functional domain comprises APOBEC3 deaminase. In some embodiments, the APOBEC3 deaminase is APOBEC3A (A3A). In some embodiments, A3A is human A3A. In some embodiments, A3A is wild-type A3A.

[0490] IX. Determination of the efficacy of guide RNA In some embodiments, the efficacy of the guide RNA when delivered or expressed together with other components forming the RNP (e.g., an RNA-guided DNA binder) is determined. In some embodiments, the guide RNA is expressed together with an RNA-guided DNA binder (such as a Cas protein, e.g., Cas9). In some embodiments, the guide RNA is delivered to or expressed in cell lines that have stably expressed RNA-guided DNA nucleases (such as Cas nucleases or nicking enzymes, e.g., Cas9 nucleases or nicking enzymes). In some embodiments, the guide RNA is delivered to cells as part of the RNP. In some embodiments, the guide RNA is delivered to cells together with mRNA encoding an RNA-guided DNA nuclease (such as Cas nucleases or nicking enzymes, e.g., Cas9 nucleases or nicking enzymes).

[0491] As described herein, the use of RNA-guided DNA nucleases and guide RNAs disclosed herein can induce DSB, SSB, or site-specific binding, leading to nucleic acid modifications in DNA or pre-mRNA that can result in insertion / deletion mutations after repair by cellular mechanisms. Many insertion / deletion mutations alter reading frames, introduce premature stop codons, or induce exon skipping, thus producing non-functional proteins.

[0492] In some embodiments, the efficacy of a specific guide RNA is determined based on an in vitro model. In some embodiments, the in vitro model is a T cell line. In some embodiments, the in vitro model is HEK293 T cells. In some embodiments, the in vitro model is HEK293 cells stably expressing Cas9 (HEK293_Cas9). In some embodiments, the in vitro model is a lymphoblastoid cell line. In some embodiments, the in vitro model is primary human T cells. In some embodiments, the in vitro model is primary human B cells. In some embodiments, the in vitro model is primary human peripheral blood lymphocytes. In some embodiments, the in vitro model is primary human peripheral blood mononuclear cells.

[0493] In some embodiments, the number of off-target sites where deletions or insertions occur in an in vitro model is determined, for example, by analyzing genomic DNA from cells in vitro transfected with Cas9 mRNA and guide RNA. In some embodiments, this determination includes analyzing genomic DNA from cells in vitro transfected with Cas9 mRNA, guide RNA, and donor oligonucleotides. Exemplary procedures for such determination are provided in the working examples below.

[0494] In some embodiments, the efficacy of a specific gRNA is determined in multiple in vitro cell models used in the guide RNA selection process. In some embodiments, data are compared with cell lines containing the selected guide RNA. In some embodiments, cross-screening is performed in multiple cell models.

[0495] In some implementations, the efficacy of the guide RNA is evaluated by target cleavage efficiency. In some implementations, the efficacy of the guide RNA is measured by the percentage of editing at the target site. In some implementations, deep sequencing can be used to identify the presence of modifications (e.g., insertions, deletions) introduced through gene editing. The insertion / deletion percentage can be calculated from next-generation sequencing (NGS).

[0496] In some embodiments, the efficacy of the guide RNA is measured by the number or frequency of insertions / deletions at off-target sequences within the genome of the target cell type. In some embodiments, effective guide RNAs are provided that produce insertions / deletions at off-target sites at a very low frequency (e.g., <5%) in the cell population or relative to the insertion / deletion generation frequency at the target site. Therefore, this disclosure provides guide RNAs that do not exhibit off-target insertion / deletion formation in the target cell type (e.g., T cells or B cells) or produce an off-target insertion / deletion formation frequency of <5% in the cell population or relative to the insertion / deletion generation frequency at the target site. In some embodiments, this disclosure provides guide RNAs that do not exhibit any off-target insertion / deletion formation in the target cell type (e.g., T cells or B cells). In some embodiments, guide RNAs that produce insertions / deletions at fewer than 5 off-target sites, for example, as evaluated by one or more methods described herein. In some embodiments, guide RNAs that produce insertions / deletions at fewer than or equal to 4, 3, 2, or 1 off-target sites, for example, as evaluated by one or more methods described herein. In some implementations, one or more off-target sites do not appear in the protein-coding regions of the genome of the target cell (e.g., T cells or B cells).

[0497] In some implementations, linear amplification is used to detect gene editing events, such as the formation of insertion / deletion (“indel”) mutations, translocations, and homology-directed repair (HDR) events in the target DNA. For example, linear amplification can be performed using primers with unique sequence markers, and the marked amplification products can be isolated (hereinafter referred to as the “UnIT” or “Unique Identifier Marking” method).

[0498] In some embodiments, the efficacy of the guide RNA is measured by the number of chromosomal rearrangements within the target cell type. Chromosomal rearrangements can be detected using the Kramitid dGH assay, including, for example, translocations, reciprocal translocations, translocations to off-target chromosomes, and deletions (i.e., chromosomal rearrangements in which segments are lost due to editing events during the cell replication cycle). In some embodiments, the target cell type has fewer than 10, 8, 5, 4, 3, 2, or 1 chromosomal rearrangements. In some embodiments, the target cell type has no chromosomal rearrangements.

[0499] X. Delivery of the composition Lipid nanoparticles (LNPs) are a well-known method for delivering nucleotide and protein cargoes and can be used to deliver the guide RNAs, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, LNP compositions deliver nucleic acids, proteins, or nucleic acids together with proteins.

[0500] In some embodiments, this disclosure provides a method for delivering any of the gRNAs disclosed herein to a subject, wherein the gRNA is formulated as an LNP. In some embodiments, the LNP comprises the gRNA and Cas9 or mRNA encoding Cas9.

[0501] In some embodiments, this disclosure provides a composition comprising any of the disclosed gRNAs and an LNP. In some embodiments, the composition further comprises Cas9 or mRNA encoding Cas9.

[0502] In some embodiments, the LNP composition comprises a cationic lipid. In some embodiments, the LNP composition comprises octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadecano-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, or another ionizable lipid. See, for example, lipids as described in WO / 2017 / 173054 and the references described therein. In some embodiments, the LNP composition comprises a cationic lipid amine to RNA phosphate (N:P) molar ratio of about 4.5, 5.0, 5.5, 6.0, or 6.5. In some implementations, the terms cationic and ionizable are interchangeable in the case of LNP lipids, for example, where the ionizable lipid is cationic depending on the pH.

[0503] In some embodiments, the LNP comprises a lipid component, and said lipid component comprises: about 35 mol% lipid A; about 15 mol% neutral lipids (e.g., distearylphosphatidylcholine (DSPC)); about 47.5 mol% accessory lipids (e.g., cholesterol); and about 2.5 mol% occult lipids (e.g., 1,2-dimyristoyl-racemic-glycerol-3-methylpolyoxyethylene glycol 2000 (PEG2k-DMG)), and wherein the N / P ratio of the LNP composition is about 3-7.

[0504] In some embodiments, the LNP comprises a lipid component, and said lipid component comprises an ionizable lipid (octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadecano-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester), cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50% ionizable lipid, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG.

[0505] In some embodiments, the gRNA disclosed herein is formulated as an LNP composition for use in the preparation of a medicament for treating a disease or condition.

[0506] Electroporation is a well-known method for delivering cargo, and any electroporation method can be used to deliver any of the gRNAs disclosed herein. In some embodiments, electroporation can be used to deliver any of the gRNAs disclosed herein and Cas9 or mRNA encoding Cas9.

[0507] In some embodiments, this disclosure includes a method for delivering any of the gRNAs disclosed herein to ex vivo cells, wherein the gRNA is formulated as an LNP or not formulated as an LNP. In some embodiments, the LNP comprises the gRNA and Cas9 or mRNA encoding Cas9.

[0508] In some embodiments, the guide RNA composition described herein, encoded alone or on one or more vectors, is formulated in or administered via lipid nanoparticles; see, for example, WO / 2017 / 173054 and WO2019 / 067992, the contents of which are hereby incorporated in their entirety by reference.

[0509] In some embodiments, this disclosure includes a DNA or RNA vector encoding any one of the guide RNAs comprising any one or more of the guide sequences described herein. In some embodiments, in addition to the guide RNA sequence, the vector also includes nucleic acids that do not encode guide RNA. Nucleic acids that do not encode guide RNA include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding RNA-guided DNA nucleases, such as Cas9. In some embodiments, the vector includes one or more nucleotide sequences encoding crRNA, trRNA, or a combination of crRNA and trRNA. In some embodiments, the vector includes one or more nucleotide sequences encoding sgRNA and mRNA encoding an RNA-guided DNA nuclease (which may be a Cas nuclease, such as Cas9). In some embodiments, the vector includes one or more nucleotide sequences encoding crRNA, trRNA, and mRNA encoding an RNA-guided DNA nuclease (which may be a Cas protein, such as Cas9). In one embodiment, the Cas9 nuclease is derived from Neisseria meningitidis (i.e., Nme Cas9). In some embodiments, the nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA (which may be sgRNA) includes or is composed of a guide sequence, said guide sequence being side-joined by all or part of a repetitive sequence from a naturally occurring CRISPR / Cas system. The nucleic acid containing crRNA, trRNA, or crRNA and trRNA, or composed of them, may also include a vector sequence, wherein the vector sequence contains or is composed of a nucleic acid not naturally found with crRNA, trRNA, or crRNA and trRNA.

[0510] In some embodiments, the component may be introduced into cells as naked nucleic acid, as nucleic acid conjugated with an agent such as liposomes or poloxamer, or the component may be delivered via a viral vector (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus). Methods and compositions for non-viral delivery of nucleic acids include electroporation, lipid transfection, microinjection, gene gun, virions, liposomes, immunoliposomes, LNPs, polycationic or lipid:nucleic acid conjugates, naked nucleic acids (e.g., naked DNA / RNA), artificial viral particles, and agent-enhanced DNA uptake. Ultrasonic perforation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used for nucleic acid delivery.

[0511] A. Exemplary cell types In some embodiments, the methods and compositions disclosed herein genetically modify cells. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are human cells. In some embodiments, the genetically modified cells are referred to as engineered cells. An engineered cell refers to a cell (or a progeny of a cell) containing engineered genetic modifications, for example, cells that have been contacted with and genetically modified by a genome editing system. The terms “engineered cell” and “genetically modified cell” are used interchangeably throughout this document. An engineered cell can be any of the exemplary cell types disclosed herein.

[0512] In some embodiments, the cells are immune cells. As used herein, "immune cell" refers to cells of the immune system, including, for example, lymphocytes (e.g., T cells, B cells, natural killer cells ("NK cells" and NKT cells or iNKT cells)), monocytes, macrophages, basophils, dendritic cells, or granulocytes (e.g., neutrophils, eosinophils, and basophils). In some embodiments, the cells are primary immune cells. In some embodiments, immune system cells may be selected from CD3+ cells. + CD4 + and CD8 + T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DCs). In some implementations, the immune cells are allogeneic.

[0513] In some embodiments, the cells are lymphocytes. In some embodiments, the cells are adaptive immune cells. In some embodiments, the cells are T cells. In some embodiments, the cells are B cells. In some embodiments, the cells are NK cells. In some embodiments, the lymphocytes are allogeneic.

[0514] As used herein, a T cell can be defined as a cell that expresses the T cell receptor (“TCR”, “αβ TCR”, or “γδ TCR”). However, in some embodiments, the TCR of T cells can be genetically modified to reduce its expression (e.g., by genetic modification of the TRAC or TRBC genes), so the expression of the protein CD3 can be used as a marker for identifying T cells using standard flow cytometry methods. CD3 is a multi-unit signal transduction complex associated with the TCR. Therefore, T cells can be referred to as CD3+. In some embodiments, T cells are cells that express both CD3+ and CD4+ or CD8+ markers. In some embodiments, T cells are allogeneic.

[0515] In some implementations, T cells express the glycoprotein CD8 and are therefore CD8+ by standard flow cytometry, and can be referred to as “cytotoxic” T cells. In some implementations, T cells express the glycoprotein CD4 and are therefore CD4+ by standard flow cytometry, and can be referred to as “helper” T cells. CD4+ T cells can differentiate into subgroups and can be referred to as Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, regulatory T cells (“Treg”), or follicular helper T cells (“Tfh”). Each CD4+ subgroup releases specific cytokines that can have pro-inflammatory or anti-inflammatory functions, or survival or protective functions. T cells can be isolated from a subject using CD4+ or CD8+ selection methods.

[0516] In some implementations, the T cells are memory T cells. In the body, memory T cells have encountered antigens. Memory T cells can be located in secondary lymphoid organs (central memory T cells) or recently infected tissues (effective memory T cells). Memory T cells can be CD8+ T cells. Memory T cells can be CD4+ T cells.

[0517] As used in this article, “central memory T cells” can be defined as T cells that have experienced antigens and, for example, can express CD62L and CD45RO. Central memory T cells can be detected as CD62L+ and CD45RO+, and central memory T cells also express CCR7, so they can be detected as CCR7+ using standard flow cytometry methods.

[0518] As used in this article, “early stem cell memory T cells” (or “Tscm”) can be defined as T cells expressing CD27 and CD45RA, and therefore, by standard flow cytometry, they are CD27+ and CD45RA+. Tscm do not express the CD45 isoform CD45RO, and therefore, if stained for this isoform by standard flow cytometry, Tscm will further be CD45RO-. Therefore, CD45RO- CD27+ cells are also early stem cell memory T cells. Tscm cells further express CD62L and CCR7, and therefore can be detected by standard flow cytometry as CD62L+ and CCR7+. Early stem cell memory T cells have been shown to be associated with increased durability and therapeutic efficacy of cell therapy products.

[0519] In some embodiments, the cells are B cells. As used herein, “B cell” can be defined as a cell that expresses CD19 or CD20 or B cell maturation antigen (“BCMA”), and therefore, by standard flow cytometry, the B cell is CD19+, CD20+, or BCMA+. By standard flow cytometry, the B cell is further CD3 and CD56 negative. The B cell can be a plasma cell. The B cell can be a memory B cell. The B cell can be a primary B cell. The B cell can be IgM+, or have class-switching B cell receptors (e.g., IgG+ or IgA+). In some embodiments, the B cell is allogeneic.

[0520] In some embodiments, the cells are monocytes, such as those derived from bone marrow or peripheral blood. In some embodiments, the cells are peripheral blood monocytes (“PBMCs”). In some embodiments, the cells are PBMCs, such as lymphocytes or monocytes. In some embodiments, the cells are peripheral blood lymphocytes (“PBLs”). In some embodiments, the monocytes are allogeneic.

[0521] This includes cells used in ACTs or tissue regeneration therapies, such as stem cells, progenitor cells, and primary cells. Stem cells include, for example, pluripotent stem cells (PSCs); induced pluripotent stem cells (iPSCs); embryonic stem cells (ESCs); mesenchymal stem cells (MSCs, such as those isolated from bone marrow (BM), peripheral blood (PB), placenta, umbilical cord (UC), or adipose tissue); hematopoietic stem cells (HSCs; such as those isolated from BM or UC); neural stem cells (NSCs); tissue-specific progenitor stem cells (TSPSCs); and limbal stem cells (LSCs). Progenitor cells and primary cells include monocytes (MNCs, such as those isolated from BM or PB); endothelial progenitor cells (EPCs, such as those isolated from BM, PB, and UC); neural progenitor cells (NPCs); and tissue-specific primary cells or cells derived therefrom (TSCs), including chondrocytes, myocytes, and keratinocytes. It also includes cells used for organ or tissue transplantation, such as pancreatic islet cells, cardiomyocytes, thyroid cells, thymocytes, neurons, skin cells, and retinal cells.

[0522] In some embodiments, the cells are human cells, such as cells isolated from human subjects. In some embodiments, the cells are isolated from human donor PBMCs or leukopaks. In some embodiments, the cells are derived from subjects suffering from diseases, ailments, or illnesses. In some embodiments, the cells are derived from human donors possessing Epstein Barr Virus ("EBV").

[0523] In some embodiments, the method is performed ex vivo. As used herein, "ex vivo" refers to an in vitro method in which cells can be transferred to a subject, for example, as an ACT therapy. In some embodiments, the ex vivo method is an in vitro method involving ACT therapy cells or cell populations.

[0524] In some embodiments, the cells are derived from a cell line. In some embodiments, the cell line is derived from a human subject. In some embodiments, the cell line is a lymphoblastoid cell line (“LCL”). The cells can be cryopreserved and thawed. The cells may not have been previously cryopreserved.

[0525] In some embodiments, the cells are derived from a cell bank. In some embodiments, the cells are genetically modified and then transferred to a cell bank. In some embodiments, cells are taken from a subject, genetically modified in vitro, and then transferred to a cell bank. In some embodiments, a population of genetically modified cells is transferred to a cell bank. In some embodiments, a population of genetically modified immune cells is transferred to a cell bank. In some embodiments, a population of genetically modified immune cells is transferred to a cell bank, said population comprising first and second subpopulations, wherein the first and second subpopulations have at least one common genetic modification and at least one different genetic modification.

[0526] B. Treatment methods and uses Any of the engineered cells and compositions described herein can be used in methods for treating a variety of diseases and conditions as described herein. In some embodiments, genetically modified cells (engineered cells) or populations of genetically modified cells (engineered cells) and compositions can be used in methods for treating a variety of diseases and conditions. In some embodiments, a method for treating any of the diseases or conditions described herein is covered, the method comprising administering any one or more compositions described herein.

[0527] In some embodiments, this disclosure provides a method for treating a subject in need, the method comprising administering engineered cells prepared by a cell preparation method described herein, such as the foregoing aspects of cell preparation methods and any of the methods described in the embodiments.

[0528] In some embodiments, the methods and compositions described herein can be used to treat diseases or conditions that require the delivery of therapeutic agents.

[0529] In some embodiments, the method includes administering to a subject a composition comprising engineered cells as described herein, wherein said cells produce, secrete, or express polypeptides (e.g., targeting receptors) that can be used to treat a disease or condition of the subject. In some embodiments, the cells act as cell factories to produce soluble polypeptides. In some embodiments, the cells act as cell factories to produce antibodies. In some embodiments, the cells continuously secrete polypeptides in vivo. In some embodiments, the cells continuously secrete polypeptides for at least 1, 2, 3, 4, 5, or 6 weeks after in vivo transplantation. In some embodiments, the cells continuously secrete polypeptides for more than 6 weeks after in vivo transplantation. In some embodiments, the soluble polypeptide (e.g., antibody) is administered at a dose of at least 10 mg / day. 2 10 3 10 4 10 5 10 6 10 7 Or 10 8A concentration of one copy is produced by the cells. In some implementations, the peptide is an antibody and is produced at a concentration of at least 10 copies per day. 8 The concentration of one copy is produced by the cell.

[0530] In some embodiments, this disclosure provides a method for preparing engineered cells (e.g., engineered cell populations). Engineered cell populations can be used in immunotherapy.

[0531] In some embodiments, this disclosure provides a method of delivering immunotherapy to a subject, the method comprising administering to the subject an effective amount of engineered cells (or a population of engineered cells) as described herein, such as cells from any of the foregoing cell aspects and embodiments.

[0532] Immunotherapy treats disease by activating or suppressing the immune system. Immunotherapy designed to trigger or amplify an immune response is classified as activating immunotherapy. Cell-based immunotherapy has proven effective in treating some cancers. Immune effector cells, such as lymphocytes, macrophages, dendritic cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), T helper cells, B cells, or their progenitor cells, such as hematopoietic stem cells (HSCs) or induced pluripotent stem cells (iPSCs), can be programmed to respond to abnormal antigens expressed on the surface of tumor cells. Therefore, cancer immunotherapy allows components of the immune system to destroy tumors or other cancer cells.

[0533] Cell-based immunotherapy has also proven effective in treating autoimmune diseases or transplant rejection. Immune effector cells, such as regulatory T cells (Tregs) or mesenchymal stem cells, can be programmed to respond to self-antigens or transplant antigens expressed on the surface of normal tissues. Immunotherapy can also be used to treat chronic infectious diseases such as hepatitis B and hepatitis C virus infections, human immunodeficiency virus (HIV) infection, tuberculosis infection, and malaria infection. Immune effector cells containing targeted receptors (such as transgenic TCRs or CARs) can be used in immunotherapies, such as those described in this article.

[0534] While transient TGFBR2 expression plays a crucial role in promoting normal immune responses, chronic TGFBR2 expression is associated with T cell exhaustion, which is the response of T cells to chronic antigen stimulation (van Gisbergen et al. 2009; Yang et al. 2014). Therefore, in some embodiments, this disclosure provides improved methods and compositions for enhancing immune responses by reducing chronic TGFBR2-mediated aberrant immune responses, such as T cell exhaustion.

[0535] In some embodiments, the engineered cell population exhibits increased expansion compared to an unmodified cell population expressing TGFBR2. In some embodiments, the cell population exhibits reduced exhaustion compared to an unmodified cell population expressing TGFBR2. In some embodiments, the cell population exhibits an increased percentage of stem cell-like memory T cells (Tscm) compared to an unmodified cell population expressing TGFBR2.

[0536] In some embodiments, the cell population exhibits increased durability compared to an unmodified cell population expressing TGFBR2. In some embodiments, the cell population exhibits increased persistence compared to an unmodified cell population expressing TGFBR2. In some embodiments, the cell population exhibits reduced cannibalism compared to an unmodified cell population expressing TGFBR2. In some embodiments, the cell population exhibits increased cytotoxicity compared to an unmodified cell population expressing TGFBR2. In some embodiments, the cell population exhibits reduced tumor volume compared to an unmodified cell population expressing TGFBR2. In some embodiments, the cell population results in reduced cancer cell area compared to an unmodified cell population expressing TGFBR2. In some embodiments, the cell population results in increased tumor clearance compared to an unmodified cell population expressing TGFBR2.

[0537] In another aspect, this disclosure provides a method for preparing cells (e.g., cell populations) for immunotherapy, the method comprising: (a) modifying cells by reducing or eliminating the expression of the TGFBR2 protein, for example by introducing a gRNA molecule (as described herein) into the cells, as disclosed herein; and (b) amplifying the cells.

[0538] In another aspect, this disclosure provides a method for preparing cells (e.g., cell populations) for immunotherapy, the method comprising: (a) modifying the cells by reducing or eliminating the expression of TGFBR2, for example by introducing a gRNA molecule (as described herein) into the cells, or by reducing or eliminating the expression of one or more components of a T cell receptor, or by introducing more than one gRNA molecule into the cells, as disclosed herein; and (b) amplifying the cells.

[0539] The cells of this disclosure are suitable for further engineering, for example by introducing a heterologous sequence encoding a target receptor, such as a polypeptide mediating TCR / CD3 ζ chain signaling. In some embodiments, the polypeptide is a target receptor selected from non-endogenous TCR or CAR sequences. In some embodiments, the polypeptide is a wild-type or variant TCR. The cells according to this disclosure are also suitable for further engineering by introducing a heterologous sequence encoding an alternative antigen-binding moiety, for example by introducing a heterologous sequence encoding an alternative (non-endogenous) T cell receptor, such as a chimeric antigen receptor (CAR) engineered to target a specific protein. CARs are also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors.

[0540] In some embodiments, the method includes administering to a subject a composition comprising the engineered cells described herein as an adoptive cell transfer therapy. In some embodiments, the engineered cells are allogeneic cells.

[0541] In some embodiments of the method, the method includes administering a lymphocyte scavenger or an immunosuppressant prior to administering an effective amount of engineered cells (or a variety of engineered cells) as described herein, such as cells from any of the foregoing cell aspects and embodiments. In another aspect, the present invention provides a method for preparing engineered cells (e.g., a population of engineered cells).

[0542] In some implementations, engineered cells can be used to treat cancer, infectious diseases, inflammatory diseases, autoimmune diseases, cardiovascular diseases, neurological diseases, ophthalmic diseases, kidney diseases, liver diseases, musculoskeletal diseases, erythrocyte disorders, or transplant rejection. In some implementations, engineered cells can be used for cell transplantation, such as transplantation into the heart, liver, lungs, kidneys, pancreas, skin, or brain. (See, for example, Deuse et al., Nature Biotechnology 37:252-258 (2019).) In some embodiments, engineered cells can be used as cell therapies including allogeneic stem cell therapy. In some embodiments, the cell therapy includes induced pluripotent stem cells (iPSCs). iPSCs can be induced to differentiate into other cell types, including, for example, cardiomyocytes, β-islet cells, neurons, and blood cells. In some embodiments, the cell therapy includes hematopoietic stem cells. In some embodiments, the allogeneic stem cell graft includes an allogeneic bone marrow graft. In some embodiments, the stem cells comprise pluripotent stem cells (PSCs). In some embodiments, the stem cells include induced embryonic stem cells (ESCs).

[0543] The engineered cells disclosed herein are suitable for further engineering, for example by introducing further edited or modified genes or alleles. The cells of this invention are also suitable for further engineering by introducing exogenous nucleic acids encoding, for example, target receptors (e.g., TCRs, CARs, UniCARs). CARs are also known as chimeric immune receptors, chimeric T-cell receptors, or artificial T-cell receptors. In some embodiments, the TCR is a wild-type or variant TCR.

[0544] In some embodiments, the cell therapy is a transgenic T-cell therapy. In some embodiments, the cell therapy includes transgenic T cells targeting Wilms' tumor 1 (WT1). In some embodiments, the cell therapy includes a donor nucleic acid that targets a receptor or encodes a target receptor for commercially available T-cell therapies (such as CAR T-cell therapy). Many target receptors are currently approved for use in cell therapies. The cells and methods described herein can be used with these known constructs. Commercially approved cell products that include target receptor constructs for use in cell therapies include, for example, Kymriah® (tisagenlecleucel); Yescarta® (axicabtagene ciloleucel); Tecartus™ (brexucabtagene autoleucel); Tab-cel® (Tabelecleucel); Viraliym-M (ALVR105); and Viraliym-C.

[0545] In some embodiments, the method provides administering engineered cells to a subject, wherein the administration is by injection. In some embodiments, the method provides administering engineered cells to a subject, wherein the administration is by intravenous injection or infusion. In some embodiments, the method provides administering engineered cells to a subject, wherein the administration is a single dose.

[0546] In some embodiments, the method provides a reduction in signs or symptoms associated with a disease in a subject treated with the compositions disclosed herein. In some embodiments, the subject has a response to treatment with the compositions disclosed herein that lasts for more than one week. In some embodiments, the subject has a response to treatment with the compositions disclosed herein that lasts for more than two weeks. In some embodiments, the subject has a response to treatment with the compositions disclosed herein that lasts for more than three weeks. In some embodiments, the subject has a response to treatment with the compositions disclosed herein that lasts for more than one month.

[0547] In some embodiments, the method provides the administration of engineered cells to a subject, wherein the subject responds to the administered cells, the response including a reduction in signs or symptoms associated with a disease treated with cell therapy. In some embodiments, the subject has a response lasting longer than one week. In some embodiments, the subject has a response lasting longer than one month. In some embodiments, the subject has a response lasting at least 1-6 weeks.

[0548] This description and exemplary embodiments should not be considered limiting. For the purposes of this specification and the appended claims, unless otherwise indicated, all figures and other numerical values ​​expressing quantities, percentages, or proportions used in this specification and the claims should be understood to be modified by the term "about" in all cases (to the extent that it is not so modified). Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and the appended claims are approximations that may vary depending on the desired characteristics sought and tolerances acceptable in the art. To a minimum, and not to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted according to the number of significant figures reported and the application of conventional rounding techniques.

[0549] References: Table 10. Extra Sequences Table 10A. Additional NME guide RNA sequences

[0550] Table 10B. Additional SPY Guide RNA Sequences

[0551] *The guide sequences disclosed in this table may be unmodified, modified using the exemplary modification patterns shown in the table, or modified using different modification patterns disclosed herein or available in the art. Throughout this application, the terms "mA", "mC", "mU", or "mG" may be used to denote a nucleotide modified with 2'-O-Me. Throughout this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide linked to the next (e.g., 3') nucleotide via a phosphate thioester (PS) bond.

[0552] XI. Examples The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of this disclosure in any way.

[0553] Example 1: General Method Generally, unless otherwise indicated, the guide RNA labeled “GXXXXXX” used throughout the embodiments refers to a modified sgRNA format, such as those shown in the tables provided herein.

[0554] 1.1 In vitro transcription of mRNA ("IVT") Using a linearized plasmid DNA template and T7 RNA polymerase, capped and polyadenylated mRNA containing N1-methyl-pseudo-U was generated via in vitro transcription. The linearized plasmid DNA containing the T7 promoter and transcription sequence was linearized by restriction endonuclease digestion, followed by heat inactivation of the reaction mixture and purification from the enzyme and buffer salts. Messenger RNA was synthesized and purified using standard techniques known in the art.

[0555] Messenger RNA was generated from plasmid DNA encoding an open reading frame, according to the sequences included in Supplementary Sequence Listing 10. When the sequence is referred to hereinafter as mRNA, it should be understood that T should be replaced by U (e.g., N1-methylpseuuridine as described above). The messenger RNA used in the examples comprised a 5' cap and a 3' polyadenylated region, for example, up to 100 nt. The guide RNA was chemically synthesized using methods known in the art.

[0556] 1.2 T cell preparation T cells were isolated from commercially obtained donor blood cells using apheresis and then cryopreserved. After thawing, the T cells were cultured at 1.0 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 100 cells / mL in T cell growth medium (TCGM), which consisted of CTSOpTmizer T cell expansion SFM and T cell expansion supplement (ThermoFisher catalog number A1048501). The supplement contained 5% human AB serum (GeminiBio, catalog number 100-512), 1X penicillin-streptomycin (ThermoFisher, 15140122), 1X Glutamax (ThermoFisher, 35050061), and 10 mM HEPES (ThermoFisher, 15630106), and was further supplemented with 200 U / mL recombinant human interleukin-2 (Peprotech, catalog number 200-02), 5 ng / mL recombinant human interleukin-7 (Peprotech, catalog number 200-07), and 5 ng / mL recombinant human interleukin-15 (Peprotech, catalog number 200-15). Before transfection, T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec, catalog number 130-111-160).

[0557] 1.3 LNP formulations Generally, lipid components are dissolved in 100% ethanol at various molar ratios. RNA cargo (e.g., Cas9 mRNA and sgRNA) is dissolved in 25 mM citrate buffer and 100 mM NaCl (pH 5.0) to achieve a concentration of approximately 0.45 mg / mL.

[0558] Lipid nanoparticles (LNPs) contain ionizable lipid (octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadecano-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, referred to herein as lipid A. The molar ratio of lipids contained in LNPs is 35 lipid A: 47.5 cholesterol: 15 DSPC: 2.5 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (PEG2K-DMG) (e.g., catalog number GM-020, from NOF, Tokyo, Japan), in molar concentration. LNPs were prepared with a molar ratio of approximately 6 lipid amines to RNA phosphate (N:P) and a weight ratio of 1:1 gRNA to mRNA.

[0559] LNPs were prepared using a cross-flow technique that utilizes an impingement jet mixing of lipid-containing ethanol with two volumes of RNA solution and one volume of water. The lipid-containing ethanol was mixed with the two volumes of RNA solution via a cross-flow mixer. A fourth stream of water was then mixed with the outlet stream of the cross-flow mixer via an inline tee (see Figure 2, WO2016010840). The LNPs were incubated at room temperature for 1 hour and further diluted with water (approximately 1:1 v / v). The diluted LNPs were buffer-exchanged to 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) and concentrated as needed using methods known in the art. The resulting mixture was then filtered through a 0.2 μm sterile filter. The final LNPs were stored at 4°C or -80°C until further use.

[0560] 1.4 Next-generation sequencing (“NGS”) and analysis of editing efficiency.

[0561] According to the manufacturer's instructions, use a commercial kit, such as QuickExtract™ DNA Extraction Solution (Lucigen, catalog number QE09050), to extract DNA.

[0562] To quantitatively determine editing efficiency at target sites in the genome, deep sequencing is used to identify the presence of insertions, deletions, and substitutions introduced through gene editing. PCR primers are designed around the target site within the gene of interest (e.g., TGFBR2), and the genomic region of interest is amplified. Primer sequences are designed according to standards in the art. Additional PCR is performed according to manufacturer protocols (e.g., Illumina, PacBio) to add chemicals for sequencing. The amplicon is sequenced on an Illumina MiSeq instrument. After eliminating reads with low quality scores (PHRED score <20), the reads are aligned to a reference genome (e.g., hg38). Reads overlapping with the target region of interest are re-aligned to the local genomic sequence to improve alignment. The following analyses are performed to detect insertions / deletions or determine base editor activity.

[0563] 1.4.1 Insertion / Missing Data Analysis The resulting file containing reads is mapped to a reference genome (BAM file), where reads that overlap with the target region of interest are selected, and the number of wild-type reads is compared with the number of reads containing insertions or deletions (“insertions / deletions”).

[0564] Edit percentage (e.g., "edit efficiency" or "insertion / deletion percentage") is defined as the ratio of the total number of sequence reads with insertions or deletions ("insertions / deletions") to the total number of sequence reads, including wild-type reads. Insertions and deletions are scored within an approximately 20 bp region centered on the predicted Cas9 cleavage site. The insertion / deletion percentage is defined as the total number of sequencing reads with one or more bases inserted or deleted within the approximately 20 bp scoring region divided by the total number of sequencing reads, including wild-type reads.

[0565] 1.4.2 Detection of base editor activity Reads overlapping with the target region of interest were re-aligned with the local genomic sequence to improve alignment. The ratio of wild-type reads to reads containing C-to-T mutations, C-to-A / G mutations, or insertions / deletions was then calculated. Insertions and deletions were scored within an approximately 20 bp region centered on the predicted Cas9 cleavage site. The insertion / deletion percentage was defined as the total number of sequencing reads with one or more bases inserted or deleted within the approximately 20 bp scoring region divided by the total number of sequencing reads, including wild-type reads. C-to-T or C-to-A / G mutations were scored within a 40 bp region, comprising 10 bp upstream and 10 bp downstream of the 20 bp sgRNA target sequence. The C-to-T editing percentage was defined as the total number of sequencing reads with one or more C-to-T mutations within the 40 bp region divided by the total number of sequencing reads, including wild-type reads. The percentage of C-to-A / G mutations was calculated similarly.

[0566] 1.5 Flow Cytometry Phenotypic analysis of edited T cells was performed using flow cytometry to determine the expression of phosphorylated SMAD2 and SMAD3 proteins (pSMAD2 / 3). T cells were incubated at 37°C for 30 min with 10 ng / ml TGFB1 (Millipore Sigma, catalog number 11412272001). Cells were then washed and stained with live / dead stain (Thermo Fisher, catalog number L34955) and CD3-positive stain (BD Biosciences, catalog number 560176). Cells were washed again and incubated at 37°C for 10 min in 1X lysis / fixation buffer (BD Biosciences, catalog number 558049). Cells were washed with buffer and then again with HBSS (Millipore Sigma, catalog number H6648). Cells were resuspended at 4°C in 1X cold PERM buffer III (BD Biosciences, catalog number 558050) for 30 min. Cells were washed twice with FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and stained for 30 minutes at room temperature with a 1:200 dilution of pSMAD2 / 3 antibody (BDBiosciences, catalog number 562586). After incubation, cells were washed and resuspended in FACS buffer. Cells were then processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo™ software package. T cells were gated based on size, shape, viability, CD3, and pSMAD2 / 3 positivity.

[0567] Example 2. Screening TGFBR2 guide RNA using the Nme2Cas9 base editor against TGFBR2 Guide RNAs (ENSG00000XXX) were designed and their efficacy was tested using the Nme2Cas9 base editor. Guide sequences and corresponding genomic coordinates are provided (Table 2). TGFBR2 guide RNAs were screened for editing efficacy in T cells by assessing editing frequency via NGS and by evaluating the loss of intracellular expression of phosphorylated SMAD2 / 3 (pSMAD2 / 3) via flow cytometry. Phosphorylated SMAD2 / 3 is a downstream protein of TGFBR2. SMAD2 / 3 will not phosphorylate in the presence of TGFB1 if TGFBR2 is knocked out. T cells derived from single-donor hematopoiesis were prepared and activated as described in Example 1. T cells were harvested by centrifugation 72 hours after activation and were cultured at 12.5 × 10⁻⁶. 6The T cells were resuspended at a concentration of 1 T cell / mL in P3 electroporation buffer (Lonza catalog number V4SP-3960). As described in Example 2, the following electroporation mixture was used to edit T cells with sgRNA targeting the TGFBR2 locus, mRNA encoding the Nme2Cas9 base editor (SEQ ID NO: 801), and mRNA encoding UGI.

[0568] Using 1 × 10 5 An electroporation mixture was prepared by adding 1 T cell, 20 ng / µL of mRNA encoding the Nme2 base editor, 20 ng / µL of mRNA encoding UGI, and 2 μM sgRNA to a final volume of 20 µL of P3 electroporation buffer. The cells were electroporated as described in Example 2. On day 3 post-electroporation, edited T cell samples were harvested and subjected to NGS analysis as described in Example 1. On day 7 post-editing, functional TGFB signaling of the T cells was determined by flow cytometry as described in Example 1.

[0569] Table 11 and Figure 1 The mean percentage of C-to-T edits for TGFBR2, the mean percentage of reads that yielded stop codons within the frame, the mean percentage of TGFBR2-negative T cells, and the mean percentage of pSMAD2 / 3-negative T cells are shown. No samples showed C-to-G edits exceeding 2%, and no samples showed insertions / deletions exceeding 3%.

[0570] Table 11. Percentage of C-to-T editing, percentage of reads acquiring the stop codon at TGFBR2, and percentage of T cells negative for phosphorylated SMAD2 / 3.

[0571] Example 3. Dosage-response analysis of TGFBR2 guide RNA using the Nme2Cas9 base editor TGFBR2 guide RNAs were screened for editing efficacy in T cells by assessing editing frequency using NGS and evaluating the loss of intracellular expression of phosphorylated SMAD2 / 3 (pSMAD2 / 3) using flow cytometry. Phosphorylated SMAD2 / 3 is a downstream protein of TGFBR2. In TGFBR2 knockout T cells, SMAD2 / 3 is not phosphorylated in the presence of TGFB1. Dose-response assays assessed the effect of increasing guide RNA levels at fixed concentrations of mRNA encoding the Nme2Cas9 base editor and mRNA encoding UGI.

[0572] T cells obtained from commercially available healthy human donor blood cell ablation were prepared and activated as described in Example 1. Forty-eight hours after activation, the T cells were harvested by centrifugation and cultured at 12.5 × 10⁻⁶. 6 The T cells / mL concentration was then resuspended in P3 electroporation buffer (Lonza catalog number V4SP-3960). As described in Table 12, 1 × 10⁻⁶ T cells / mL were used. 5 A base editor electroporation mixture was prepared using 10 T cells, 20 ng / µL of mRNA encoding a base editor, 20 ng / µL of mRNA encoding a UGI, and sgRNA. The mixture was transferred to the corresponding wells of a 96-well Nucleofector™ plate (Lonza catalog number V4SP-3960). Using a Lonza shuttle 96w, cells were electroporated in duplicate according to the manufacturer's pulse code. Immediately after electroporation, cells were recovered in 80 µL of TCGM containing 5% human AB serum and cytokines as described in Example 1, and incubated at 37°C for 5 minutes. The electroporated T cells were then cultured with an additional 100 µL of TCGM containing 5% human AB serum and cytokines as listed in the T cell preparation. The plate was incubated at 37°C with 5% CO2. On day 3 after electroporation, the edited T cells were harvested and processed for NGS analysis as described in Example 1. On day 7 after electroporation, T cells were phenotypically analyzed by flow cytometry.

[0573] Table 12 and Figure 2A-2B The average percentage of TGFBR2 edited, expressed as a percentage of total NGS reads, and the average percentage of pSMAD2 / 3 negative cells are shown.

[0574] Table 12. Mean percentage of TGFBR2 editing and mean percentage of pSMAD2 / 3 negative cells.

[0575] Example 3: Multiple editing of allogeneic CD70 CAR-T cells via LNP delivery Allogeneic anti-CD70 CAR-T cells were engineered to achieve efficient multiplexed knockout of HLA-A, HLA-B, CIITA, CD70, TGFBR2, and TRAC using LNP delivery of the editing component. Additionally, insertion of the anti-CD70 CAR into the TRAC locus was achieved by transducing a homologous targeted repair template delivered along with AAV.

[0576] 3.1 T cell preparation T cells were isolated and cryopreserved using methods known in the art. On the day prior to initiating T cell editing (day -0), CD4 and CD8 T cells were thawed, pooled at a 1:1 ratio, and incubated overnight in the following T cell activation medium (TCAM): CTS OpTmizer (Thermofisher, catalog number A3705001) supplemented with 2.5% human AB serum (ValleyBiomedical HP1022HI), 1x GlutaMAX (ThermoFisher 35050061), 10 mM HEPES (ThermoFisher 15630080), 100 U / mL Penstrep (Gibco 15140-122), 200 U / mL IL-2 (Peprotech 200-02), IL-7 (Peprotech 200-07), and IL-15 (Peprotech 200-15). Biological replication experiments were performed using T cells isolated from three donors.

[0577] 3.2 LNP treatment and T cell expansion Day 1, T cells were harvested and at a rate of 1×10 6 Cells were resuspended at a density of cells / mL in TCAM containing a 1:100 dilution of TransAct (Miltenyi, 130-111-160). Cells were treated with LNPs and AAV as described in Table 13. Treatments included 10 μg / mL ApoE3 (Peprotech, catalog number 350-02) on days 1 and 3, and a DNA protein kinase inhibitor (such as DNAPKI compound 1 disclosed herein) on day 3. LNPs were generally prepared as described in Example 1. LNPs were prepared with a molar ratio of lipid amines to RNA phosphates (N:P) of approximately 6. LNPs with a co-formulation of gRNA and mRNA were prepared using a 1:1 gRNA to mRNA ratio by weight. The lipid nanoparticles in this example were prepared with a molar ratio of 35 lipid A: 47.5 cholesterol: 15 DSPC: 2.5 PEG2k-DMG. LNP dosage is reported as the mass of total RNA cargo per volume. The cells were incubated at 37°C until day 4.

[0578] Table 13 - Editing Schedule

[0579] On day 4, the cells were mixed, counted, and TCAM was added to adjust the cell density to 0.5 × 10⁻⁶. 6 Cells / mL, then incubated for 24 hours.

[0580] On day 5, T cells were transferred and incubated in T cell expansion medium (TCEM): supplemented with 5% CTS immune cell serum substitute (ThermoFisher A2596101), 1x GlutaMAX (Thermofisher, catalog number 35050061), 10 mM HEPES (ThermoFisher 15630080), 100 U / mL Penstrep (Gibco 15140-122), 200 U / mL IL-2 (Peprotech, catalog number 200-02), IL-7 (Peprotech, catalog number 200-07), and IL-15 (Peprotech, catalog number 200-15) in CTS OpTmizer (ThermoFisher A3705001).

[0581] Cells were expanded at 37°C on days 5-11. Cell counts were collected daily. After expansion, cells were harvested and counted using an NC200 Nucleocounter device (Chemometec) to determine cell viability and fold expansion.

[0582] For flow cytometry analysis, cells were washed and incubated in a mixture of antibodies targeting the following substances: CD4 (BioLegend 317434), CD8a (BioLegend 301046), CD3 (BioLegend 300430), HLA-A2 (BioLegend 343320), HLA-A3 (BD ​​Biosciences 747776), HLA-B7 (Miltenyi Biotec 130-120-234), HLA-A9 (Miltenyi Biotec 130-099-539), HLA-DR / DP / DQ (BioLegend 361712) as surrogate readings for CIITA function, HLA-Bw4 (Miltenyi Biotec 130-103-851), CD45RA (BioLegend 304126), CD62L ...04126), and CD62L (BioLegend 304126). CAR insertion was detected using labeled anti-Fc (BioLegend 410708) and CD70-Fc (Sino Biological 10780-H01H) as primary antibodies, and viability was determined using VioKrome live / dead dye (Beckman Coulter C36628). Flow cytometry data were acquired on a Cytoflex LX instrument (Beckman Coulter) and analyzed using FlowJo software.

[0583] Table 14 and Figure 3 Surface protein expression and cell viability were shown as detected by flow cytometry. CD3 is an alternative marker for TRAC editing. HLA-DR / DQ / DP- indicates successful disruption of CIITA. HLA-A- and HLA-B- status were reported using antibodies appropriate for the donor genotype. Tscm (CD45RA+, CD62L+) and Tcm (CD45RA-, CD62L+) indicate the memory cell population. Table 15 and Figure 3 This shows C to T edits at TGFBR2 or CD70, in Figure 3 The results were reported as "TGFBR2-" or "CD70-". The percentage of fully edited allogeneic-CD70 CAR T cells was estimated to be an average of approximately 54% by multiplying the percentage of HLA-A-, HLA-B-, CD3-, and CAR+ cells by the C-to-T editing percentage at TGFBR2 and CD70.

[0584] Table 14 shows the percentage of engineered cells with a specified cell surface phenotype, as determined by flow cytometry.

[0585] Table 15. Percentage of C-to-T editing in engineered cells via NGS.

[0586] References:

Claims

1. An engineered cell, said engineered cell comprising gene modifications within genomic coordinates chr3:30606891-30691605.

2. An engineered cell having reduced or eliminated surface expression of TGFβR2 protein relative to unmodified cells, and containing gene modifications within genomic coordinates chr3:30606891-30691605.

3. The engineered cell of claim 1 or 2, wherein the gene modification is within genomic coordinates targeted by a guide RNA, the guide RNA comprising a guide sequence of any one of SEQ ID NO: 1-49.

4. The engineered cell according to any one of claims 1-3, wherein the engineered cell has reduced or eliminated TGFβR2 surface expression relative to the unmodified cell, and contains gene modifications within any of the genomic coordinates listed in Table 2.

5. The engineered cell according to any one of claims 1-4, wherein the gene modification is within a genomic coordinate system selected from: chr3:30674205-30674229;chr3:30671674-30671698;chr3:30671677-30671701;chr3:30674167-30674191;chr3:30672133-30672157;chr3:30606891-30606915;chr3:30606892-30606916;chr3:30606896-30606920;chr3:30606897-30606921;chr3:30606898-30606922;chr3:30606899-30606923;chr3:30606908-30606932;chr3:30606909-30606933;chr3:30606910-30606934;chr3:30606917-30606941;chr3:30606958-30606982;chr3:30606959-30606983;chr3:30606960-30606984;chr3:30606964-30606988;chr3:30606965-30606989;chr3:30644900-30644924;chr3:30671667-30671691;chr3:30671670-30671694;chr3:30671753-30671777;chr3:30671762-30671786;chr3:30671766-30671790;chr3:30672034-30672058;chr3:30672126-30672150;chr3:30672128-30672152;chr3:30672131-30672155;chr3:30672135-30672159;chr3:30672139-30672163;chr3:30672140-30672164;chr3:30672140-30672164;chr3:30672141-30672165;chr3:30672190-30672214;chr3:30672204-30672228;chr3:30672432-30672456;chr3:30672433-30672457;chr3:30672434-30672458;chr3:30674211-30674235;chr3:30688450-30688474;chr3:30688459-30688483;chr3:30688460-30688484; chr3:30688476-30688500; chr3:30688478-30688502; chr3:30688479-30688503; chr3:30691436-30691460; and chr3:30691581-30691605.

6. The engineered cell of any one of claims 1-5, wherein the gene modification is within genomic coordinates targeted by the guide RNA, the guide RNA comprising a guide sequence of any one of SEQ ID NO: 1-5.

7. The engineered cell according to any one of claims 1-6, wherein the gene modification is within the genomic coordinates selected from the following: chr3:30674205-30674229; chr3:30671674-30671698; chr3:30671677-30671701; chr3:30674167-30674191; and chr3:30672133-30672157.

8. The engineered cell of any one of claims 1-7, wherein the gene modification is within genomic coordinates targeted by a guide RNA, the guide RNA comprising the guide sequence of SEQ ID NO:

1.

9. The engineered cell according to any one of claims 1-8, wherein the gene modification is located within the genomic coordinates chr3:30674205-30674229.

10. A composition comprising a guide RNA and optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the guide RNA comprises (a) A guide sequence selected from SEQ ID NO: 1-49; (b) A guide sequence of at least 20, 21, 22, 23, 24 or 25 consecutive nucleotides selected from the sequences of SEQ ID NO: 1-49; (c) A guide sequence having at least 85%, 90%, or 95% identity with a sequence selected from SEQ ID NO: 1-49; (d) A sequence containing 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; (e) At least 20, 21, 22, 23, or 24 consecutive nucleotides from the sequence in (d); or (f) A guide sequence that has at least 85%, 90% or 95% identity with a sequence selected from (d).

11. The composition of claim 10, wherein the composition is used to alter the DNA sequence within the TGFβR2 gene in a cell.

12. A pharmaceutical composition comprising the composition of claim 10, or the use of the composition of claim 10 for inducing double-strand breaks or single-strand breaks in the TGFβR2 gene in cells, altering the nucleic acid sequence of the TGFβR2 gene in cells, or reducing the expression of the TGFβR2 gene in cells.

13. A method for manufacturing engineered human cells having reduced or eliminated surface expression of TGFβR2 protein relative to unmodified cells, the method comprising contacting the cells with the composition of claim 10.

14. A method for reducing the surface expression of TGFβR2 protein in cells relative to unmodified cells, the method comprising contacting the cells with a composition comprising a guide RNA and optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the guide RNA comprises a. A guide sequence selected from SEQ ID NO: 1-49; b. A guide sequence of at least 20, 21, 22, 23, 24 or 25 consecutive nucleotides selected from the sequence of SEQ ID NO: 1-49; c. A guide sequence having at least 85%, 90%, or 95% identity with a sequence selected from SEQ ID NO: 1-49; d. A sequence containing 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; e. At least 20, 21, 22, 23, 24, or 25 consecutive nucleotides from the sequence in (d); or f. A guide sequence that has at least 85%, 90%, or 95% identity with a sequence selected from (d).

15. The composition, use, or method of any one of claims 10-14, wherein the guide RNA comprises the guide sequence of SEQ ID NO:

1.

16. The composition, use, or method of any one of claims 10-15, wherein the RNA-guided DNA binder is a base editor.

17. A cell population comprising engineered cells produced by using a composition as described in any one of claims 10-12, 15 and 16 or a method as described in any one of claims 13-16.

18. A pharmaceutical composition comprising (a) engineered cells produced by any one of the compositions or methods of claims 10-16; or (b) a cell population as described in claim 17.

19. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 1-18, wherein the gene modification comprises insertion, deletion, or substitution.

20. The engineered cell, cell population, pharmaceutical composition, or method according to any one of claims 1-19, wherein the gene modification comprises insertion / deletion, C-to-T substitution, or A-to-G substitution within the genomic coordinates.

21. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 1-20, wherein the cells are engineered using a genome editing system.

22. The engineered cells, cell populations, pharmaceutical compositions or methods according to any one of claims 3-21, wherein the guide RNA is a dual guide RNA (dgRNA) or a single guide RNA (sgRNA).

23. The engineered cells, cell populations, pharmaceutical compositions, or methods of claim 22, wherein the sgRNA is an Nme sgRNA comprising a guide region and a conserved region.

24. The engineered cells, cell populations, pharmaceutical compositions, or methods of claim 23, wherein the conserved region comprises one or more of the following: (a) A shortened repeat / anti-repeat region, wherein the shortened repeat / anti-repeat region is missing 2-24 nucleotides relative to SEQ ID NO: 700, wherein (i) One or more of nucleotides 37-48 and 53-64 are deleted relative to SEQ ID NO: 700, and optionally one or more of nucleotides 37-64 are substituted relative to SEQ ID NO: 700; and (ii) Nucleotide 36 is composed of at least two nucleotides linked to nucleotide 65; or (b) The shortened hairpin 1 region, wherein the shortened hairpin 1 is missing 2-10 nucleotides, optionally 2-8 nucleotides, relative to SEQ ID NO: 700, wherein (i) One or more of nucleotides 82-86 and 91-95 are deleted relative to SEQ ID NO: 700, and optionally one or more of positions 82-96 are substituted relative to SEQ ID NO: 700; and (ii) Nucleotide 81 is linked to nucleotide 96 by at least four nucleotides; or (c) The shortened hairpin 2 region, wherein the shortened hairpin 2 is missing 2-18 nucleotides, optionally 2-16 nucleotides, relative to SEQ ID NO: 700, wherein (i) One or more of nucleotides 113-121 and 126-134 are deleted relative to SEQ ID NO: 700, and optionally one or more of nucleotides 113-134 are substituted relative to SEQ ID NO: 700; and (ii) Nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides; One or both of nucleotides 144-145 are optionally deleted relative to SEQ ID NO: 700; Optionally, at least 10 of the nucleotides are modified nucleotides.

25. The engineered cell, cell population, pharmaceutical composition, or method of claim 23 or 24, wherein the conserved region comprises a modified nucleotide sequence selected from the modified conserved region Nme guide RNA motifs in Table 6, and wherein the conserved region is located at the 3' of the guide region.

26. The engineered cell, cell population, pharmaceutical composition, or method of claim 23, wherein the guide RNA comprises a nucleotide sequence selected from any of SEQ ID NO: 701-706, and wherein N represents a guide sequence selected from any of SEQ ID NO: 1-49.

27. The engineered cells, cell populations, pharmaceutical compositions, or methods of claim 26, wherein each nucleotide is any natural or non-natural nucleotide, and / or wherein the guide RNA is modified according to the sequence or modification pattern listed in Tables 6-7, wherein N is the guide sequence, N, A, C, G, and U are ribonucleotides (2'-OH), "m" indicates 2'-O-Me modification, "f" indicates 2'-fluorine modification, and "*" indicates phosphate thioester linkage between nucleotides.

28. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 3-27, wherein the guide RNA comprises at least one end modification, optionally wherein the modification comprises a 5' end modification and / or wherein the modification comprises a 3' end modification.

29. The engineered cells, cell populations, pharmaceutical compositions, or methods of claim 28, wherein the guide RNA comprises a modification in the hairpin region, optionally wherein the modification in the hairpin region is also an end modification.

30. The engineered cells, cell populations, pharmaceutical compositions, or methods of claim 28 or 29, wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide, and / or wherein the modification comprises a phosphate thioester (PS) bond between nucleotides, and / or wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide linked to a 3' adjacent nucleotide by a phosphate thioester (PS) bond, and / or wherein the modification comprises a 2'-fluorine (2'F) modified nucleotide, and / or wherein the 5' end modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide at positions 1-3 of the 5' end of the guide sequence linked to a 3' adjacent nucleotide by a phosphate thioester (PS) bond.

31. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 3-30, wherein the guide RNA is associated with lipid nanoparticles (LNPs), optionally wherein the LNPs comprise cationic lipids, helper lipids, neutral lipids, stealth lipids, or a combination of two or more thereof.

32. The engineered cells, cell populations, pharmaceutical compositions, or methods of claim 31, wherein the cationic lipid is octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadecano-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, and / or wherein the auxiliary lipid is cholesterol, and / or wherein the neutral lipid is 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), and / or wherein the occult lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000. (PEG2k-DMG), and / or said LNP comprises octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octoxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadecano-9,12-dienoic acid 3-((4,4-bis(octoxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, DSPC, cholesterol, and PEG2k-DMG.

33. A pharmaceutical composition comprising engineered cells as described in any one of claims 1-32.

34. A cell population comprising engineered cells as described in any one of claims 1-32.

35. A pharmaceutical composition comprising a cell population, wherein the cell population comprises a plurality of engineered cells as described in any one of claims 1-32, and optionally wherein the pharmaceutical composition further comprises a pharmaceutical excipient.

36. A method of administering engineered cells, cell populations, or pharmaceutical compositions as described in any one of claims 1-35 to a subject in need, wherein the engineered cells, cell populations, or pharmaceutical compositions are administered to the subject as adoptive cell transfer (ACT) therapy or as immunotherapy.

37. The engineered cells, cell populations, or pharmaceutical compositions according to any one of claims 1-35, wherein the engineered cells, cell populations, or pharmaceutical compositions are used as ACT therapy.

38. A method of treating a disease or condition, the method comprising administering to a subject in need engineered cells, cell populations or pharmaceutical compositions as described in any one of claims 1-35.

39. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 1-38, wherein the engineered cells have reduced surface expression of TGFβR2 protein compared to unmodified cells.

40. The engineered cell, cell population, pharmaceutical composition, or method according to any one of claims 1-39, wherein the cell comprises an exogenous nucleic acid encoding a target receptor expressed on the surface of the engineered cell, optionally wherein the target receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

41. The engineered cells, cell populations, pharmaceutical compositions, or cell methods of claim 40, wherein the targeted receptor is WT1 TCR.

42. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 1-41, wherein the engineered cells further comprise gene modifications of one or more of the CIITA, HLA-A, HLA-B, or TRAC genes, and / or wherein the engineered cells further have reduced surface expression of one or more of the MHC class II proteins, HLA-A, HLA-B, or TRAC compared to unmodified cells.

43. The engineered cells, cell populations, pharmaceutical compositions, or methods of claim 42, wherein the engineered cells comprise: i. Gene modifications within the genomic coordinates chr6:29942891-29942915 or chr6:29942609-29942633 of the HLA-A gene; ii. Gene modifications in the HLA-B gene selected from the genomic coordinates of chr6:31355222-31355246, chr6:31355221-31355245 and chr6:31355205-31355229; iii. Gene modifications in the TRAC gene selected from the genomic coordinates of chr14:22547524-22547544, chr14:22550574-22550598 and chr14:22550544-22550568; iv. Gene modifications within the genomic coordinates chr16:10906643-10906667 or chr16:10907504-10907528 of the CIITA gene; or v. A combination of two or more items from (i)-(iv).

44. The engineered cells, cell populations, pharmaceutical compositions, or methods of claim 42 or 43, wherein the engineered cells comprise at least one genetic modification, said genetic modification being: (i) within genomic coordinates targeted by an HLA-A guide RNA containing a guide sequence of SEQ ID NO: 403 or 404; (ii) within genomic coordinates targeted by an HLA-B guide RNA containing a guide sequence of SEQ ID NO: 406, 405, or 407; (iii) within genomic coordinates targeted by a TRAC guide RNA containing a guide sequence of SEQ ID NO: 413, 408, or 409; (iv) within genomic coordinates targeted by a CIITA guide RNA containing a guide sequence of SEQ ID NO: 402 or 401; or (v) a combination of two or more of (i)-(iv).

45. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 42-44, wherein the engineered cells comprise gene modifications in the HLA-A gene, gene modifications in the HLA-B gene, gene modifications in the TRAC gene, and gene modifications in the CIITA gene.

46. ​​The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 42-45, wherein the engineered cells comprise: i. Gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene; ii. Gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene; iii. Gene modifications within the genomic coordinates chr14:22547524-22547544 of the TRAC gene; and iv. Gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene.

47. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 42-46, wherein the engineered cells comprise: i. Gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene; ii. Gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene; iii. Gene modifications within the genomic coordinates chr14:22547524-22547544 of the TRAC gene; iv. Gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene; and v. Gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFβR2 gene.

48. An engineered human cell, said engineered human cell comprising gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFβR2 gene, and gene modifications within the genomic coordinates chr14:22547524-22547544 of the TRAC gene.

49. The engineered cell, cell population, pharmaceutical composition, or method according to any one of claims 1-48, wherein the engineered cell is an immune cell, and optionally wherein the engineered cell is a lymphocyte.

50. The engineered cell, cell population, pharmaceutical composition, or method of claim 49, wherein the engineered cell is a T cell, optionally wherein the cell is a CD4+ T cell or a CD8+ T cell, and / or wherein the cell is a memory T cell.

51. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 1-50, wherein the cells are allogeneic cells.

52. The engineered cells, cell populations, pharmaceutical compositions, or methods according to any one of claims 1-51, wherein the engineered cells, cell populations, pharmaceutical compositions, or methods are administered to a subject as an adoptive cell transfer (ACT) therapy, for treating a subject with cancer, for treating a subject with an infectious disease, or for treating a subject with an autoimmune disease.

53. The population or pharmaceutical composition of any one of claims 17-52, wherein, as measured by flow cytometry, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cell population is TGFβR2 negative.

54. The population or pharmaceutical composition of any one of claims 17-53, wherein, as measured by next-generation sequencing (NGS), at least 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cell population contains the genetic modification in the TGFβR2 gene.

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