HK40116920A - Reduced expression of sarm1 for use in cell therapy - Google Patents

Description

Reduced SARM1 expression is used in cell therapy.

This application claims the benefit of U.S. Provisional Application No. 63/284,995, filed December 1, 2021, the contents of which are incorporated herein by reference.

Throughout this application, various publications have been referenced, including those in parentheses. The disclosures of all publications mentioned herein are incorporated herein by reference in their entirety to provide additional description of the field to which this invention pertains and the features that may be employed in this field.

References to sequence lists

This application incorporates by reference the nucleotide sequence contained in a file named “221201_91818-A-PCT_Sequence_Listing_AWG.xml”, which is 11,660 kilobytes in size, created on December 1, 2022, in IBM-PC machine format and compatible with the MS-Windows operating system, and is included in the XML file filed on December 1, 2022, as part of this application.

Background Technology

Immunotherapy, such as CAR T-cell therapy, offers promising avenues for treating several diseases and conditions. However, to make such therapies more effective, there is a strong desire to develop allogeneic adoptive transfer strategies in which engineered cells exhibit increased persistence after transfer to a patient.

Summary of the Invention

The sterile α and TIR motif 1 (SARM1) gene is an NAD+ hydrolase that acts as a negative regulator of the MYD88- and TRIF-dependent Toll-like receptor signaling pathway by promoting Wallerian degeneration (a damage-induced form of programmed subcellular death). SARM1 can also activate cell death in response to stress (Molday et al., 2013).

Methods for knocking out, knocking down, or inhibiting SARM1 gene expression in engineered immune cell types to increase its persistence, proliferation, and/or retention during cell therapy are disclosed. Therefore, biallelic knockout, knockdown, or inhibition of SARM1 gene expression in cell therapy-related cell types as described herein can be used to improve cell therapies, including for the treatment of cancer.

This disclosure also provides methods for adoptive cell therapy or prevention, comprising administering SARM1-inhibited or SARM1-inactivated cells to an individual suffering from or identified as being at risk of developing cancer, infection, disease, or condition. In some embodiments, the cells are immune cells (e.g., lymphocytes, monocytes, or macrophages). In some embodiments, the SARM1-inhibited immune cells are autologous. In other embodiments, the SARM1-inhibited immune cells are allogeneic.

Attached Figure Description

Figures 1A-1B: Screening for RNA guide molecule activity targeting SARM1 in HeLa cells. Cells were harvested 72 hours after DNA transfection. Genomic DNA was extracted and subjected to capillary electrophoresis after amplification of endogenous genomic regions using on-target primers. The figures represent the mean ± standard deviation (STDV) of the edit percentage from three (3) independent experiments. Figure 1A: Co-transfection of SpCas9-encoding plasmids with plasmids expressing the RNA guide molecule shown. Figure 1B: Co-transfection of OMNI-50 or OMNI-79 CRISPR nucleases with the RNA guide molecule shown.

Figure 2: Electroporation of the RNP of the SpCas9 nuclease complexed with a specific RNA guide molecule into Neuro-2a cells to determine RNP activity. Cells were harvested 72 hours after DNA electroporation, and genomic DNA was extracted and then analyzed by next-generation sequencing (NGS). This figure represents the edit % ± STDV of two (2) independent electroporations.

Figure 3: Relative amounts of edited SARM1 RNA. Neuro-2a cells were harvested seven (7) days after electroporation, and RNA was extracted and reverse transcribed. The relative amounts of SARM1 RNA were quantified using the AriaMx system. The level of mRNA was quantified relative to unedited, untreated cells.

Figure 4: Editing activity of the OMNI-103CRISPR nuclease with an RNA guide molecule targeting SARM1 in HeLa cells. Specific RNA guide molecules were co-transfected with the OMNI-103CRISPR nuclease to determine their targeting activity. Cells were harvested 72 hours after DNA transfection, genomic DNA was extracted, the mutated region was amplified, and analyzed by NGS. Transfection efficiency was measured by mCherry fluorescence. This figure represents the editing % ± STDV of three (3) independent transfections.

Figure 5: SARM1 pathway diagram.

Figure 6A: SARM1 editing and NGS results. Figure 6B: Kill test. Figure 6C: Viability (ATPlite) test.

Detailed Implementation

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials described herein may be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials will be described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to impose necessary limitations.

It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to the components listed as “one or more”. Those skilled in the art will appreciate that the use of the singular includes the plural unless otherwise specifically stated. Therefore, the terms “a,” “an,” and “at least one” are used interchangeably in this application.

To better understand this teaching, and in no way to limit its scope, all figures and other numerical values used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximate values that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based on the number of significant figures disclosed and by applying common rounding techniques.

Unless otherwise stated, adjectives such as “substantially” and “about” modifying a conditional or relational feature of one or more features of an embodiment of the invention are understood to mean that the condition or feature is limited to an operationally acceptable tolerance range for the intended application of that embodiment. Unless otherwise stated, the word “or” in the specification and claims is considered inclusive rather than exclusive and indicates at least one or any combination of the terms.

In the specification and claims of this application, each verb "comprising," "including," and "having," and their variations, are used to indicate that one or more objects of the verb do not necessarily fully enumerate the components, elements, or parts of one or more subjects of the verb. As used herein, other terms are intended to be defined by their meanings well known in the art.

In some embodiments of the invention, DNA nucleases are used to influence DNA breaks at target sites to induce cellular repair mechanisms, such as, but not limited to, non-homologous end joining (NHEJ). In a typical NHEJ process, the two ends of a double-strand break (DSB) site join together rapidly but inaccurately (i.e., often resulting in mutations in the DNA at the cleavage site as small insertions or deletions).

As used herein, the term "modified cell" refers to a cell in which double-strand breaks are affected by the complex of RNA molecules and CRISPR nucleases due to hybridization with the target sequence, i.e., mid-target hybridization.

As used herein, the term "target sequence" or "target molecule" refers to a nucleotide sequence or molecule that contains a nucleotide sequence capable of hybridizing with a specific target sequence, for example, a target sequence having a nucleotide sequence at least partially complementary to the sequence targeted along the length of the target sequence. The target sequence or target molecule can be part of an RNA molecule that can form a complex with a CRISPR nuclease alone, or in combination with other RNA molecules, wherein the target sequence serves as the targeting portion of the CRISPR complex. When a molecule having a target sequence is present in conjunction with a CRISPR molecule, the RNA molecule, alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule), can target the CRISPR nuclease to a specific target sequence. As a non-limiting example, the guide sequence portion of a CRISPR RNA molecule or a single guide RNA molecule can serve as a target molecule. Each possibility represents a separate implementation. The target sequence can be custom-designed to target any desired sequence.

As used herein, the term "targeting" refers to the preferential hybridization of the target sequence of a target molecule with a nucleic acid having the targeted nucleotide sequence. It should be understood that the term "targeting" includes varying hybridization efficiencies, thus preferentially targeting nucleic acids having the targeted nucleotide sequence; however, unintentional off-target hybridization may also occur in addition to on-target hybridization. It should be understood that when an RNA molecule targets a sequence, the complex of the RNA molecule and the CRISPR nuclease molecule targets that sequence to achieve nuclease activity.

The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence capable of hybridizing with a specific target DNA sequence. For example, the guide sequence portion has nucleotide sequences that are either completely complementary to or aligned with a DNA sequence portion targeted along its length. In some embodiments, the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, 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, or 50 nucleotides long, or approximately 17-50, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17-42, 17-41, 17-40, or 17... -39, 17-38, 17-37, 17-36, 17-35, 17-34, 17-33, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-22, 17-21, 18-25, 18-24, 18-23, 18-22, 18-21, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-22, 18-20, 20-21, 21-22, or 17-20 nucleotides. The full length of the guide sequence is perfectly complementary to the DNA sequence targeted along the length of the guide sequence. The guide sequence portion can be a part of an RNA molecule that can form a complex with a CRISPR nuclease, serving as the DNA targeting portion of the CRISPR complex. When an RNA molecule with a guide sequence portion is present in conjunction with a CRISPR molecule, either alone or in combination with one or more other RNA molecules (e.g., tracrRNA molecules), the RNA molecule is capable of targeting the CRISPR nuclease to a specific target DNA sequence. Therefore, a CRISPR complex can be formed by the direct binding of an RNA molecule with a guide sequence portion to a CRISPR nuclease or by the binding of an RNA molecule with a guide sequence portion and one or more other RNA molecules to a CRISPR nuclease. Each possibility represents a separate implementation. The guide sequence portion can be custom-designed to target any desired sequence. Therefore, a molecule containing a “guide sequence portion” is a targeting molecule. In some implementations, the guide sequence portion comprises a sequence that is identical to or differs from the guide sequence portion described herein by no more than 1, 2, 3, 4, or 5 nucleotides, such as the guide sequence shown in any of SEQ ID NO: 1-13457. Each possibility represents a separate implementation. In some of these embodiments, the guide sequence portion comprises a sequence identical to that shown in any of SEQ ID NO:1-13457. In this application, the terms "guide molecule," "RNA guide molecule," "guide RNA molecule," and "gRNA molecule" are synonymous with "molecule containing a guide sequence portion."

The term “non-discriminatory” as used in this article refers to a specific DNA sequence common to all alleles of a gene targeted by the guide sequence portion of an RNA molecule.

In embodiments of the invention, the RNA molecule targeting SARM1 comprises a guide sequence portion having 17-50 consecutive nucleotides containing nucleotides of the sequences shown in any one of SEQ ID NO: 1-13457. The RNA molecule and/or the guide sequence portion of the RNA molecule may contain modified nucleotides. Exemplary modifications to nucleotides/polynucleotides may be synthetic and cover polynucleotides containing nucleotides other than naturally occurring adenine, cytosine, thymine, uracil, or guanine bases. Modifications to polynucleotides include polynucleotides containing synthetic, non-naturally occurring nucleosides, such as locked nucleic acids. Modifications to polynucleotides can be used to increase or decrease the stability of RNA. An example of a modified polynucleotide is mRNA containing 1-methylpseuuridine. Examples of modified polynucleotides and their uses can be found in U.S. Patent 8,278,036, PCT International Publication No. WO/2015/006747, and Weissman and Kariko (2015), each of which is incorporated herein by reference.

As used herein, “continuous nucleotide” as shown in SEQ ID NO means a nucleotide sequence arranged in the order shown in SEQ ID NO without any inserted nucleotides.

In embodiments of the present invention, the guide sequence portion of the RNA molecule targeting SARM1 may be 50 nucleotides in length and contain 20-22 consecutive nucleotides of the sequence shown in any one of SEQ ID NO: 1-13457. In embodiments of the present invention, the guide sequence portion may be less than 22 nucleotides in length. For example, in embodiments of the present invention, the guide sequence portion may be 17, 18, 19, 20, or 21 nucleotides in length. In such embodiments, the guide sequence portion may consist of 17, 18, 19, 20, or 21 nucleotides of the 17-22 consecutive nucleotide sequences shown in any one of SEQ ID NO: 1-13457. For example, a guide sequence portion having 17 nucleotides of the 17 consecutive nucleotide sequence shown in SEQ ID NO: 13458 may consist of any one of the following nucleotide sequences (nucleotides excluded from the consecutive sequences are marked with strikethrough):

AAAAAAAUGUACUUGGUUCC(SEQ ID NO:13458)

17-nucleotide guide sequence 1: AAAAAAAUGUACUUGGUUCC (SEQ ID NO: 13459)

17-nucleotide guide sequence 2: AAAAAAAUGUACUUGGUUCC (SEQ ID NO: 13460)

17-nucleotide guide sequence 3: AAAAAAAUGUACUUGGUUCC (SEQ ID NO: 13461)

17-nucleotide guide sequence 4: AAAAAAAUGUACUUGGUUCC (SEQ ID NO: 13462)

In embodiments of the invention, the guide sequence portion may be longer than 20 nucleotides. For example, in embodiments of the invention, the guide sequence portion may be 21, 22, 23, 24, or 25 nucleotides long. In such embodiments, the guide sequence portion comprises 17-50 nucleotides, containing 20, 21, or 22 consecutive nucleotide sequences as shown in any of SEQ ID NO: 1-13457, and additional nucleotides that are completely complementary to the nucleotide or nucleotide sequence at the 3' end, 5' end, or both of the adjacent target sequence.

In embodiments of the invention, a CRISPR nuclease forms a CRISPR complex with an RNA molecule containing a guide sequence portion, which binds to a target DNA sequence to influence the cleavage of the target DNA sequence. A CRISPR nuclease (e.g., Cpf1) may form a CRISPR complex containing both the CRISPR nuclease and an RNA molecule, without a separate tracrRNA molecule. Alternatively, a CRISPR nuclease (e.g., Cas9) may form a CRISPR complex between the CRISPR nuclease, the RNA molecule, and the tracrRNA molecule. The guide sequence portion, containing a nucleotide sequence capable of hybridizing to a specific target DNA sequence, and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA sequence portion) may be located on the same RNA molecule. Alternatively, the guide sequence portion may be located on one RNA molecule, while the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA portion) may be located on separate RNA molecules. A single RNA molecule containing a guide sequence portion (e.g., a DNA-targeting RNA sequence) and at least one CRISPR protein-binding RNA sequence portion (e.g., a tracrRNA sequence portion) can form a complex with a CRISPR nuclease and serve as a DNA-targeting molecule. In some embodiments, a first RNA molecule containing a DNA-targeting RNA portion interacts with a second RNA molecule containing a CRISPR protein-binding RNA sequence through base pairing to form an RNA complex that targets the CRISPR nuclease to a DNA target site; alternatively, they may be fused together to form an RNA molecule that complexes with the CRISPR nuclease and targets the CRISPR nuclease to a DNA target site.

In embodiments of the invention, the RNA molecule containing the guide sequence portion may further contain the sequence of a tracrRNA molecule. Such embodiments can be designed as synthetic fusions of the guide portion of the RNA molecule and trans-activating crRNA (tracrRNA) (see Jinek et al., 2012). In such embodiments, the RNA molecule is a single guide RNA (sgRNA) molecule. Embodiments of the invention can also utilize a separate tracrRNA molecule and a separate RNA molecule containing the guide sequence portion to form a CRISPR complex. In such embodiments, the tracrRNA molecule can hybridize with the RNA molecule via base pairing and may be advantageous in certain applications of the invention described herein.

The term "tracr mate sequence" refers to a sequence that is fully complementary to the tracr RNA molecule in order to hybridize with the tracr RNA via base pairing and promote the formation of the CRISPR complex (see U.S. Patent No. 8,906,616). In embodiments of the present invention, the RNA molecule may further include a portion having the tracr mate sequence.

For the purposes of this invention, "gene" includes the DNA region encoding a gene product, as well as all DNA regions that regulate the production of the gene product, regardless of whether these regulatory sequences are adjacent to the coding and/or transcriptional sequences. Therefore, a gene includes, but is not limited to, promoter sequences, terminators, translation regulatory sequences (such as ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

"Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells.

As used herein, the term "nuclease" refers to an enzyme capable of cleaving the phosphodiester bond between nucleotide subunits of nucleic acids. Nucleases can be isolated or derived from natural sources. Natural sources can be any living organism. Alternatively, nucleases can be modified or synthetic proteins that retain phosphodiester bond-cleaving activity. Nucleases can be used to achieve gene modifications, such as CRISPR nucleases.

In the context of this invention, any one or combination of strategies for reducing or inactivating SARM1 expression in cells for use in cell therapy can be used.

According to embodiments of the present invention, a method for adoptive cell therapy or prevention is provided, comprising administering SARM1-inhibited or SARM1-inactivated cells to an individual suffering from cancer, infection, disease, or condition, or identified as being at risk of cancer, infection, disease, or condition.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are modified to have reduced or inactivated SARM1 expression, or cells are modified to express dominant-negative SARM1 sequence variants or dominant-negative fragments thereof.

In some embodiments, the cells are selected from hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPS cells), iPSc-derived cells, natural killer cells (NK cells), iPSc-derived NK cells (iNK cells), T cells, innate-like T cells (iT cells), natural killer T cells (NKT cells), γδ T cells, iPSc-derived T cells, inertial NKT cells (iNKT cells), iPSc-derived NKT cells, monocytes, or macrophages. In some embodiments, the progenitor cells are first modified and then differentiated to produce SARM1-suppressed or SARM1-inactivated cells. Alternatively, SARM1-suppressed or SARM1-inactivated cells can be produced by directly editing cells (e.g., primary NK cells).

In some embodiments, SARM1-inhibited or SARM1-inactivated cells exhibit increased functionality relative to corresponding cells expressing wild-type or unmodified SARM1. In some embodiments, SARM1-inhibited or SARM1-inactivated cells exhibit increased and/or enhanced viability relative to corresponding cells expressing wild-type or unmodified SARM1. In some embodiments, SARM1-inhibited or SARM1-inactivated cells exhibit enhanced in vivo functionality, such as cytotoxicity, transport, localization, persistence, and/or proliferation, relative to corresponding cells expressing wild-type or unmodified SARM1.

In some implementations, SARM1-inhibited or SARM1-inactivated cells exhibit increased functionality, increased viability, increased persistence, increased proliferation, and/or increased tumor retention in vivo compared to corresponding cells with wild-type or unmodified SARM1 expression.

In some embodiments, SARM1-inhibited or SARM1-inactivated cells exhibit increased cytotoxicity and/or increased killing activity in vivo compared to corresponding cells expressing wild-type or unmodified SARM1. For example, in some embodiments, SARM1-inhibited or SARM1-inactivated cells exhibit higher target cell killing activity compared to corresponding cells expressing wild-type or unmodified SARM1.

In some implementations, an individual has cancer or is identified as being at risk of developing cancer.

In some implementations, cancer includes tumors and/or cancer is a hematologic malignancy.

In some implementation schemes, the cancer is selected from static melanoma, metastatic prostate cancer, metastatic breast cancer, triple-negative breast cancer, bladder cancer, brain cancer, esophageal cancer, liver cancer, head and neck cancer, squamous cell lung cancer, non-small cell lung cancer, Merkel cell carcinoma, sarcoma, hepatocellular carcinoma, multiple myeloma, leukemia, non-Hodgkin lymphoma, lymphoma, B-cell lymphoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, cervical cancer, gastric cancer, kidney cancer, metastatic renal cell carcinoma, leukemia, ovarian cancer, and malignant glioma.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are generated in vitro or ex vivo.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are generated in vivo.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are generated from cells obtained from an individual through mobilization and/or leukocyte separation.

In some implementations, the cells are obtained from the individual through bone marrow aspiration.

In some implementations, cells are pre-stimulated before SARM1 inhibition or inactivation.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are cultured and expanded before being administered to an individual.

In some implementations, SARM1-inhibited or SARM1-inactivated cells can be transplanted.

In some implementations, SARM1-inhibited or SARM1-inactivated cells can generate daughter cells.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are able to produce progeny cells after transplantation.

In some implementations, SARM1-inhibited or SARM1-inactivated cells can generate progeny cells after autologous transplantation.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are able to produce progeny cells for at least 12 months or at least 24 months after transplantation.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are generated by delivering gapmers, shRNA, siRNA, custom TALENs, macronucleases, zinc finger nucleases, CRISPR nucleases, or small molecule inhibitors to the cells.

In some implementations, the alleles of the SARM1 gene in SARM1-suppressed or SARM1-inactivated cells undergo insertion or deletion mutations.

In some implementations, insertion or deletion mutations produce early stop codons.

In some implementations, SARM1-inhibited or SARM1-inactivated cells are generated by methods including the following:

Introducing a composition into cells, the composition comprising:

At least one CRISPR nuclease, or a nucleotide molecule encoding a CRISPR nuclease; and

An RNA molecule containing a guide sequence portion, or a nucleotide molecule encoding said RNA molecule.

The complex of CRISPR nuclease and RNA molecules affects double-strand breaks in the SARM1 gene alleles.

The guide sequence portion of the RNA molecule contains 17-50 consecutive nucleotides.

In some implementations, the guide sequence portion is complementary to the target sequence located 50 base pairs upstream to 50 base pairs downstream of exons I, II, III, IV, V, VI, VII, VIII, or IX of the SARM1 gene.

In some implementations, the guide sequence portion is complementary to the target sequence located 30 base pairs upstream to 30 base pairs downstream of the exon of the SARM1 gene, and

a) The exon is exon I, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 1-56, 301-356, 1348-2223, 57-114, 357-414, 2224-3095, 115-174, 415-474, 3096-3963 by no more than 3 nucleotides;

b) The exon is exon II, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO:3964-7683 by no more than 3 nucleotides;

c) The exon is exon III, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO:7684-8967 by no more than 3 nucleotides;

d) The exon is exon IV, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO:8968-9525 by no more than 3 nucleotides;

e) The exon is exon V, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO: 9526-10947 by no more than 3 nucleotides;

f) The exon is exon VI, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO: 10948-11571 by no more than 3 nucleotides;

g) The exon is exon VII, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO:11572-12717 by no more than 3 nucleotides;

h) The exon is exon VIII, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 12718-13455 by no more than 3 nucleotides; or

i) The exon is exon IX, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO:598-1347 by no more than 3 nucleotides.

In some embodiments, the guide sequence portion comprises 17-50 consecutive nucleotides, said 17-50 consecutive nucleotides containing the nucleotides in the sequences shown in any one of SEQ ID NO:1-13457.

In some implementations, SARM1-inhibited or SARM1-inactivated cells exhibit increased viability and/or increased functionality compared to corresponding cells with wild-type or unmodified SARM1 expression.

According to embodiments of the present invention, a medicament comprising SARM1-inhibited or SARM1-inactivated cells is provided for treating or preventing an individual's cancer, infection, disease, or condition according to the methods described herein. In some embodiments, the present invention provides a kit for treating or preventing an individual's cancer, infection, disease, or condition, comprising a medicament and instructions for delivering said composition to an individual suffering from or identified as being at risk of developing cancer, infection, disease, or condition.

According to an embodiment of the present invention, a method for inactivating the alleles of the sterile α and Toll/interleukin-1 receptor motif 1 (SARM1) gene in cells is provided, the method comprising:

Introducing a composition into cells, the composition comprising:

At least one CRISPR nuclease, or a nucleotide molecule encoding a CRISPR nuclease; and

An RNA molecule containing a guide sequence portion, or a nucleotide molecule encoding said RNA molecule.

The complex of CRISPR nuclease and RNA molecules affects double-strand breaks in the alleles of the SARM1 gene.

The guide sequence portion comprises 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides of the sequences shown in any one of SEQ ID NO: 1-13457, and

The cells are selected from hematopoietic stem cells (HSC), induced pluripotent stem cells (iPS cells), iPSc-derived cells, natural killer cells (NK), iPS-derived NK cells (iNK), T cells, innate-like T cells (iT), natural killer T cells (NKT), γδ T cells, iPSc-derived T cells, inertial NKT cells (iNKT), iPSc-derived NKT, monocytes, or macrophages.

According to an embodiment of the present invention, a cell modified by inactivating the allele of the SARM1 gene in a cell is provided, wherein the modified cell contains at least one inactivated SARM1 allele.

In some embodiments, the modified cells contain two inactivated SARM1 alleles. In some embodiments, the modified cells exhibit increased viability and/or increased functionality compared to corresponding cells with wild-type or unmodified SARM1 expression. In some embodiments, the increased functionality includes increased cytotoxicity and/or increased killing ability.

In some implementations, the cells are used for adoptive cell therapy or prevention.

In some implementations, adoptive cell therapy or prevention is for the treatment or prevention of an individual's cancer, infection, disease, or condition.

According to embodiments of the present invention, a method is provided for inactivating SARM1 expression in cells by delivering an RNA molecule comprising a guide sequence portion (e.g., a target sequence), said guide sequence portion comprising a nucleotide sequence that is fully or partially complementary to a target sequence, said target sequence comprising an SNP position (REF/SNP sequence) located in or near an allele of the SARM1 gene. In some embodiments, the guide sequence portion of the RNA molecule consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more than 26 nucleotides. In some embodiments, the guide sequence portion is configured to target the target sequence with a CRISPR nuclease and provide a cleavage event by the CRISPR nuclease compounded therewith, said cleavage event being selected from double-strand breaks and single-strand breaks within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides at the SARM1 target site. In some embodiments, the cleavage event enables the SARM1 gene to undergo nonsense-mediated decay. In some implementations, the RNA molecule is a guide RNA molecule, such as a crRNA molecule or a single guide RNA molecule.

In some embodiments, the target sequence of the SARM1 gene allele is altered (e.g., by introducing an NHEJ-mediated insertion/deletion (indel) (e.g., insertion or deletion)) and results in reduced or eliminated expression of the gene product encoded by the SARM1 gene allele. In some embodiments, the reduced or eliminated expression is due to nonsense-mediated mRNA decay, such as due to an immature stop codon. In some embodiments, the reduced or eliminated expression is due to expression of a truncated form of the SARM1 gene product. In some embodiments, the guide sequence portion is complementary to the target sequence containing the SNP position. In some embodiments, the SNP position is rs782593684. In some embodiments, the guide sequence portion comprises a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO:1-174 by no more than 1, 2, or 3 nucleotides. In some embodiments, the guide sequence portion comprises a sequence that is identical to or differs from the sequence shown in any of the following: SEQ ID NO: 1, 4, 8, 20, 24, 26-27, 33, 35, 37, 41, 47, 49, 51, 53, 56, 175-214, 57, 60, 64, 76, 80, 82-83, 92, 94, 98, 102, 105, 107, 109, 111, 114, 215-256, 115, 118, 122, 132, 139, 141-142, 151, 157, 161, 164-165, 167, 169, 171, 174, and 257-300. Each possibility represents a separate embodiment. In some embodiments, SNP position 17:28372349_C_CT. In some embodiments, the guide sequence portion comprises a sequence that is identical to or differs from the sequence shown in any of SEQ ID NO:301-474 by no more than 1, 2, or 3 nucleotides. In some embodiments, the guide sequence portion comprises a sequence that is identical to or differs from the sequence shown in any of the following: SEQ ID NO:301, 304, 315, 321, 326-328, 335, 338, 340, 344, 346-348, 353, 355-356, 475-513, 357, 361, 364, 368, 373, 384-385, 393, 396, 398, 402, 404-406, 411, 413-414, 514-554, 415-416, 419, 422, 424, 427, 432, 443-444, 458, 462, 464-466, 471, 473-474, and 555-597. Each possibility represents a separate implementation scheme.

According to embodiments of the present invention, an RNA molecule or sequence encoding the RNA molecule is provided, wherein the RNA molecule includes a guide sequence portion (e.g., a target sequence) comprising a nucleotide sequence that is fully or partially complementary to a target sequence located in or near the SARM1 gene. In some embodiments, the guide sequence portion is complementary to a target sequence located 30 base pairs upstream to 30 base pairs downstream of exons I, II, III, IV, V, VI, VII, VIII, or IX of the SARM1 gene. In some embodiments, the guide sequence portion is complementary to a target sequence located 50 base pairs upstream to 50 base pairs downstream of exons I, II, III, IV, V, VI, VII, VIII, or IX of the SARM1 gene. Each possibility represents a separate embodiment. In some embodiments, the target sequence of the SARM1 gene is altered (e.g., by introducing an NHEJ-mediated indel (e.g., insertion or deletion)) and results in a decrease or elimination of expression of the gene product encoded by the SARM1 gene. In some embodiments, the decrease or elimination of expression is due to nonsense-mediated mRNA decay. In some embodiments, the guide sequence portion of the RNA molecule consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more than 26 nucleotides. In some embodiments, the guide sequence portion is configured to target the target sequence with a CRISPR nuclease and provide a cleavage event via a CRISPR nuclease complexed therewith, the cleavage event being selected from 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides at the SARM1 target site. In some embodiments, the cleavage event enables the SARM1 gene to undergo nonsense-mediated decay. In some embodiments, the RNA molecule is a guide RNA molecule, such as a crRNA molecule or a single guide RNA molecule.

In some embodiments, the guide sequence portion is complementary to the target sequence located 30 base pairs upstream to 30 base pairs downstream of an exon in the SARM1 gene. In some embodiments, the guide sequence portion is complementary to the target sequence located 50 base pairs upstream to 50 base pairs downstream of an exon in the SARM1 gene. In some embodiments, the guide sequence portion is complementary to the target sequence located 7 base pairs upstream to 7 base pairs downstream of an exon in the SARM1 gene. In some embodiments, the exon is exon I, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 1-56, 301-356, 1348-2223, 57-114, 357-414, 2224-3095, 115-174, 415-474, and 3096-3963 by no more than 3 nucleotides. In some embodiments, the exon is exon II, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 3964-7683 by no more than 3 nucleotides. In some embodiments, the exon is exon III, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 7684-8967 by no more than 3 nucleotides. In some embodiments, the exon is exon IV, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 8968-9525 by no more than 3 nucleotides. In some embodiments, the exon is exon V, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 9526-10947 by no more than 3 nucleotides. In some embodiments, the exon is exon VI, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 10948-11571 by no more than 3 nucleotides. In some embodiments, the exon is exon VII, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 11572-12717 by no more than 3 nucleotides. In some embodiments, the exon is exon VIII, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 12718-13455 by no more than 3 nucleotides. In some embodiments, the exon is exon IX, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 598-1347 by no more than 3 nucleotides.

In embodiments of the invention, an RNA molecule is used to guide a CRISPR nuclease to an exon or splice site in the SARM1 allele to generate a double-strand break (DSB), leading to nucleotide insertion or deletion by inducing a fault-prone non-homologous end joining (NHEJ) mechanism and frameshift mutation in the SARM1 allele. For example, a frameshift mutation can lead to SARM1 allele inactivation or knockout by generating an early stop codon in the SARM1 allele, resulting in truncated proteins or nonsense-mediated mRNA decay of the allele transcript. In other embodiments, an RNA molecule is used to guide the CRISPR nuclease to the promoter of the SARM1 allele. CRISPR compositions for inactivating the SARM1 allele as described herein may include at least one CRISPR nuclease, an RNA molecule, and a tracrRNA molecule, all of which are simultaneously effective in an individual or cell. At least one CRISPR nuclease, RNA molecule, and tracrRNA may be delivered substantially simultaneously, or may be delivered at different times but function simultaneously. For example, this includes delivering CRISPR nucleases to an individual or cell before the RNA molecule and/or tracrRNA are substantially present in the individual or cell.

According to some aspects, SARM1-inhibited or SARM1-inactivated cells are provided. In some embodiments, the cells are immune cells (e.g., monocytes, macrophages, lymphocytes, natural killer (NK) cells, iPS-derived NK cells (iNK), T cells, innate-like T cells (iT), iPS-derived T cells, natural killer T cells (NKT), invariant NKT cells (iNKT), or iPS-derived NKT cells). In some embodiments, the SARM1-inhibited cells are stem cells, HSCs, or iPScs. Each possibility represents a separate embodiment. SARM1-inhibited or SARM1-inactivated cells (e.g., immune cells) can be generated by utilizing any suitable SARM1 inhibitor (including peptides, polypeptides, proteins, nucleases, small molecules, or polynucleotides).

In some embodiments, SARM1-suppressed or SARM1-inactivated cells are genetically modified to have reduced SARM1 activity. In some embodiments, SARM1-suppressed or SARM1-inactivated cells are derived from precursor cells that have been genetically modified to reduce SARM1 activity. In a non-limiting example, SARM1-suppressed iNK cells are derived from iPSc cells that have been genetically modified to reduce SARM1 activity, for example, by using CRISPR to knock out the SARM1 allele and further differentiating them into iNK cells.

In some embodiments, SARM1-inhibited or SARM1-inactivated cells, or cells derived therefrom, are used for cell therapy. In some embodiments, SARM1-inhibited cells, or cells derived therefrom, are used for immunotherapy.

According to some aspects of this disclosure, this document provides a method for increasing cell viability and/or cell functionality by inactivating the alleles of the sterile α and toll/interleukin-1 receptor motif 1 (SARM1) gene in cells, the method comprising introducing into cells a composition comprising: a CRISPR nuclease, or a nucleotide molecule encoding a CRISPR nuclease, and an RNA molecule comprising a guide sequence portion of 17-50 consecutive nucleotides containing nucleotides of any of the sequences shown in SEQ ID NO: 1-13457, or a nucleotide molecule encoding said RNA molecule, wherein the complex of the CRISPR nuclease and the RNA molecule affects double-strand breaks in the alleles of the SARM1 gene. Non-limiting examples of cell types that can be modified by this method include, but are not limited to, liver cells (e.g., hepatocytes), lung cells, spleen cells, pancreatic cells, colon cells, skin cells, bladder cells, eye cells, ocular cells, retinal cells, corneal cells, brain cells, esophageal cells, head cells, neck cells, ovarian cells, testicular cells, prostate cells, placental cells, epithelial cells, endothelial cells, adipocytes, kidney/renal cells, heart cells, muscle cells, blood cells (e.g., leukocytes), immune cells, central nervous system (CNS) cells, ganglion cells, and combinations thereof.

SARM1 Editing Strategy

The provided method for knocking out the SARM1 allele in cells can be used to create SARM1-inactivated cells for use in cell therapy or immunotherapy.

SARM1 editing strategies include, but are not limited to: (1) bicelestek knockout by targeting any one of exons 2-9 or a combination thereof, including flanking splice donor and acceptor sites within seven nucleotides upstream and downstream of the exon, since frameshifts of these exons result in the decay of nonfunctional truncated SARM1 protein or nonsense-mediated mutant SARM1 transcripts; and (2) truncating SARM1 protein by mediating an indel upstream of exon 1 or overlapping with the second methionine codon in the exon, which would eliminate the codon and thus prevent the restart of translation, or by targeting the exon 1-intron 1 junction to disrupt the splice donor.

CRISPR nuclease and PAM recognition

In some embodiments, the sequence-specific nuclease is selected from CRISPR nucleases or their functional variants. In some embodiments, the sequence-specific nuclease is an RNA-guided DNA nuclease. In such embodiments, the RNA sequence of the RNA-guided DNA nuclease (e.g., Cpf1) binds to all SARM1 alleles in the cell, and/or the RNA-guided DNA nuclease is directed to all SARM1 alleles in the cell. In some embodiments, the CRISPR complex does not further contain tracrRNA. In a non-limiting example, wherein the RNA-guided DNA nuclease is a CRISPR protein, at least one distinct nucleotide between the dominant SARM1 allele and the functional allele may be located within and/or near a PAM site in the region to which the RNA molecule is designed to hybridize. Those skilled in the art will understand that RNA molecules can be engineered to bind to targets selected in the genome using methods known in the art.

As used herein, the term "PAM" refers to the nucleotide sequence of target DNA located near the targeted DNA sequence and recognized by the CRISPR nuclease complex. The PAM sequence can vary depending on the nuclease characteristics. Furthermore, there are CRISPR nucleases that target almost all PAMs. In some embodiments of the invention, the CRISPR system utilizes one or more RNA molecules having a guide sequence portion to guide the CRISPR nuclease to the target DNA site via Watson-Crick base pairing between the guide sequence portion and a prespacer region sequence on the target DNA site, the prespacer region sequence being adjacent to the prespacer region sequence adjacent motif (PAM), an additional requirement for target recognition. The CRISPR nuclease then mediates cleavage of the target DNA site, creating a double-strand break within the prespacer region sequence. In one non-limiting example, the type II CRISPR system utilizes a mature crRNA:tracrna complex that guides a CRISPR nuclease (e.g., Cas9) to the target DNA via Watson-Crick base pairing between the guide sequence portion of the crRNA and the pre-spacer region sequence adjacent to the pre-spacer region sequence neighboring motif (PAM) on the target DNA. Those skilled in the art will understand that each engineered RNA molecule of the present invention is also designed such that it is associated with a target genomic DNA sequence adjacent to the pre-spacer region sequence neighboring motif (PAM), for example, a PAM that matches the sequence associated with the type of CRISPR nuclease used, such as for non-limiting examples: for *Streptococcus pyogenes* Cas9 WT (SpCAS9), the PAM is NGG or NAG, where “N” is any nucleobase; for *Staphylococcus aureus* (SaC... For SpCas9, the NCGRRT is used; for Campylobacter jejuni Cas9 WT, the NCNVRYM is used; for the SpCas9-VQR variant, the NGAN or NGNG is used; for the SpCas9-VRER variant, the NGCG is used; for the SpCas9-EQR variant, the NGAG is used; for the SpCas9-NRRH variant, the NRRH is used, where N is any nucleobase, R is A or G and H is A, C or T; for the SpCas9-NRTH variant, the NRTH is used, where N is any nucleobase, R is A or G and H is A, C or T; for SpCas9... -NRCH variant, NRCH, where N is any nucleobase, R is A or G and H is A, C or T; for the SpG variant of SpCas9, NG, where N is any nucleobase; for the SpCas9-NG variant of SpCas9, NG or NA, where N is any nucleobase; for the SpRY variant of SpCas9, NR or NRN or NYN, where N is any nucleobase, R is A or G and Y is C or T; for the Streptococcus canis Cas9 variant (ScCas9), NNG, where N is any nucleobase; for The SaKKH-Cas9 variant of Staphylococcus aureus (SaCas9) is NNNRRT, where N is any nucleobase and R is A or G; for Neisseria meningitidis (NmCas9), it is NNNGATT, where N is any nucleobase; for Alicyclobacillus acidiphilus Cas12b (AacCas12b), it is TTN, where N is any nucleobase; or for Cpfl, it is TTTV, where V is A, C, or G. The RNA molecules of this invention are each designed to bind to one or more different CRISPR nucleases to form a complex, and are designed to target a polynucleotide sequence using one or more different PAM sequences corresponding to the CRISPR nuclease used.

In some implementations, RNA-guided DNA nucleases (e.g., CRISPR nucleases) can be used to induce DNA breaks at desired locations in the cell genome, which may be double-stranded or single-stranded in nature. The most commonly used RNA-guided DNA nucleases are derived from the CRISPR system; however, the use of other RNA-guided DNA nucleases in the genome editing compositions and methods described herein is also considered. See, for example, U.S. Patent Application Publication No. 2015/0211023, the entire contents of which are incorporated herein by reference.

The CRISPR systems that can be used to implement this invention vary considerably. CRISPR systems can be type I, type II, or type III. Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas1 Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or... CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6 , Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl0, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4 and Cul966.

In some implementations, the RNA-guided DNA nuclease is a CRISPR nuclease derived from the type II CRISPR system (e.g., Cas9). CRISPR nucleases can be derived from *Streptococcus pyogenes*, *Streptococcus thermophilus*, *Streptococcus* sp., *Staphylococcus aureus*, *Neisseria meningitidis*, *Treponema denticola*, *Nocardiopsis dassonvillei*, *Streptomyces pristinaespiralis*, *Streptomyces viridochromogenes*, *Streptomyces viridochromogenes*, *Streptosporangium roseum*, and *Alicyclob*. *Bacillus acidocaldarius*, *Bacillus pseudodomycoides*, *Bacillus selenitireducens*, *Exiguobacterium sibiricum*, *Lactobacillus delbrueckii*, *Lactobacillus salivarius*, *Microscilla marina*, *Burkholderiales bacterium*, *Polaromonas naphthalenivorans*, *Polaromonas* sp., *Crocosphaera watsonii*, *Cyanothece* sp., *Microcystis aeruginosa*, *Synechococcus* sp., *Acetohalobium* *Clostridium arabaticum*, *Ammonifex degensii*, *Caldicelulosiruptor becscii*, *Candidatus Desulforudis*, *Clostridium botulinum*, *Clostridium difjicile*, *Finegoldia magna*, *Natranaerobius thermophilus*, *Pelotomaculum thermopropionicum*, *Acidithiobacillus caldus*, *Acidithiobacillus ferrooxidans*, *Allochromatium vinosum*, *Marinobacter p.*, *Nitrosococcus halophilus*, *Nitrosococcus wa* *Pseudoalteromonas haloplanktis*, *Ktedonobacter racemifer*, *Methanohalobium evestigatum*, *Anabaenavariabilis*, *Nodularia spumigena*, *Nostoc* sp., *Arthrospira maxima*, *Arthrospira platensis*, *Arthrospira sp.*, *Lyngbya* sp., *Microcoleus chthonoplastes*, *Oscillatoria* sp., *Petrotoga mobilis*, *Thermosiphoafricanus*, *Acaryochloris marina*, or any species encoding a CRISPR nuclease with a known PAM sequence. CRISPR nucleases encoded by uncultured bacteria can also be used in the context of this invention (see Burstein et al., Nature, 2017). CRISPR protein variants with known PAM sequences (e.g., SpCas9 D1135E variant, SpCas9 VQR variant, SpCas9 EQR variant, or SpCas9 VRER variant) can also be used in the context of this invention.

Therefore, RNA-guided DNA nucleases of the CRISPR system, such as Cas9 protein or modified Cas9 or Cas9 homologs or orthologs, or other RNA-guided DNA nucleases belonging to other types of CRISPR systems, such as Cpf1 and its homologs and orthologs, can be used in the compositions of the present invention. Other CRISPR nucleases, such as those described in PCT International Application Publications WO2020/223514 and WO2020/223553, the contents of which are incorporated herein by reference, may also be used.

In some embodiments, the CRIPSR nuclease may be a “functional derivative” of a naturally occurring Cas protein. A “functional derivative” of a natural sequence polypeptide is a compound having the same qualitative biological properties as the natural sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of the natural sequence and derivatives of natural sequence polypeptides and their fragments, provided they have the same biological activity as the corresponding natural sequence polypeptide. The biological activity contemplated herein is the ability of the functional derivative to hydrolyze DNA substrates into fragments. The term “derivative” includes amino acid sequence variants, covalently modified forms, and fusions of the polypeptide. Suitable derivatives of Cas polypeptides or their fragments include, but are not limited to, mutants, fusions, and covalently modified forms of Cas proteins or their fragments. Cas proteins (including Cas proteins or their fragments, and derivatives of Cas proteins or their fragments) can be obtained from cells, chemically synthesized, or by a combination of both methods. Cells may be cells that naturally produce Cas proteins, or cells that naturally produce Cas proteins and are genetically engineered to produce endogenous Cas proteins at higher expression levels, or cells that produce Cas proteins from exogenously introduced nucleic acids encoding Cas proteins that are the same as or different from endogenous Cas. In some cases, cells do not produce Cas proteins naturally, but rather through genetic engineering to produce them.

In some implementations, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-guided endonuclease that utilizes a T-rich prespacer sequence adjacent to a motif. Cpf1 cleaves DNA via staggered double-strand breaks. Two Cpf1 enzymes from the genera *Acidaminococcus* and *Lachnospiraceae* have been shown to perform efficient genome editing in human cells (see Zetsche et al., 2015).

Therefore, RNA-guided DNA nucleases of type II CRISPR systems, such as Cas9 protein or modified Cas9 or Cas9 homologs, orthologs or variants, or other RNA-guided DNA nucleases belonging to other types of CRISPR systems, such as Cpf1 and its homologs, orthologs or variants, can be used in this invention.

In some embodiments, the guide molecule comprises one or more chemical modifications that impart new or improved properties (e.g., improved degradation stability, improved hybridization energy, or improved binding properties to RNA-guided DNA nucleases). Suitable chemical modifications include, but are not limited to, modified bases, modified sugar moieties, or modified internucleotide linkages. Non-limiting examples of suitable chemical modifications include: 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, β,D-galactosylqueuosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, and 1-methylpseudouridine. 1-Methylguanosine, 1-Methylinosine, "2,2-Dimethylguanosine", 2-Methyladenosine, 2-Methylguanosine, 3-Methylcytidine, 5-Methylcytidine, N6-Methyladenosine, 7-Methylguanosine, 5-Methylaminomethyluridine, 5-Methoxyaminomethyl-2-thiouridine, "β,D-Mannosylpiperidine", 5-Methoxycarbonylmethyl-2-thiouridine, 5-Methoxycarbonylmethyluridine, 5-Methoxyuridine, 2-Methylthio-N6-isopentenyl Adenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine, N-((9-β-D-ribofuranosyl-6-yl)N-methylcarbamoyl)threonine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, wybutoxosine, queuosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine 5-Methyluridine, N-((9-β-D-ribofuranopurine-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, "3-(3-amino-3-carboxypropyl)uridine,(acp3)u", 2'-O-methyl (M), 3'-thiophosphate (MS), 3'-thioPACE (MSP), pseudouridine, or 1-methylpseudouridine. Each possibility represents a separate embodiment of the invention.

In addition to targeting the SARM1 allele via RNA-guided CRISPR nucleases, other means of inhibiting SARM1 expression in target cells for cell therapy (e.g., hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPS cells), natural killer cells (NK cells), iPSC-derived NK cells (iNK), T cells, innate-like T cells (iT), γδT cells, iPSC-derived T cells, natural killer T cells (NKT), inertial NKT cells (iNKT), iPSC-derived NKT, monocytes, or macrophages) include, but are not limited to, the use of gapmers, shRNA, siRNA, custom TALENs, macronucleases or zinc finger nucleases, small molecule inhibitors, and any other methods known in the art for reducing or eliminating gene expression in target cells. See, for example, U.S. Patent Nos. 6,506,559; 7,560,438; 8,420,391; 8,552,171; 7,056,704; 7,078,196; 8,362,231; 8,372,968; 9,045,754; and PCT International Publication Nos. WO/2004/067736; WO/2006/097853 The entire contents of WO/2003/087341; WO/2000/0415661; WO/2003/080809; WO/2010/079430; WO/2010/079430; WO/2011/072246; WO/2018/057989; and WO/2017/164230 are incorporated herein by reference.

Advantageously, when complexed with CRISPR nucleases in cells, guide RNA molecules containing at least one guide sequence portion presented herein provide improved SARM1 knockout efficiency compared to other guide RNA molecules. These specially designed sequences can also be used to identify SARM1 target sites for other nucleotide-targeted gene editing or gene silencing methods, such as siRNA, TALEN, macronucleases, or zinc finger nucleases.

Delivery to cells

Any of the compositions described herein for reducing or eliminating SARM1 expression can be delivered to target cells by any suitable method. The RNA guide molecule for targeting SARM1 in the methods of this invention can be delivered to any cell intended for application to an individual for cell therapy purposes, or to generate such cells, and said cells contain and/or express the SARM1 allele. For example, in one embodiment of the invention, an RNA molecule specifically targeting the SARM1 allele is delivered to target cells, and said target cells are hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPS cells), natural killer cells (NK cells), iPS-derived NK cells (iNK cells), T cells, innate-like T cells (iT), natural killer T cells (NKT), inertial NKT cells (iNKT), monocytes, or macrophages. Delivery to cells can be performed in vivo, in vitro, or ex vivo. In some embodiments, delivery to cells is in vitro or ex vivo, so that the resulting modified cells can be applied to an individual in need. In some embodiments, delivery to cells is in vivo, so that the modification of the cells occurs in the individual in need. Furthermore, the nucleic acid compositions described herein can be delivered to cells as one or more of DNA molecules, RNA molecules, ribonucleoproteins (RNPs), nucleic acid vectors, or any combination thereof.

In some embodiments, the RNA molecule contains chemical modifications. Non-limiting examples of suitable chemical modifications include 2'-O-methyl (M), 2'-O-methyl, 3'-thiophosphate (MS), or 2'-O-methyl, 3'-thioPACE (MSP), pseudouridine, and 1-methylpseudouridine. Each possibility represents a single embodiment of the invention.

Any suitable viral vector system can be used to deliver nucleic acid compositions, such as the RNA molecular compositions of the present invention. Conventional virus- and non-viral gene transfer methods can be used to introduce nucleic acids into target tissues. In some embodiments, nucleic acids are administered for in vivo or in vitro gene therapy purposes. Non-viral vector delivery systems include naked nucleic acids as well as nucleic acids compounded with a delivery vector (such as liposomes or poloxamer). For a review of gene therapy procedures, see Anderson (1992); Nabel and Felgner (1993); Mitani and Caskey (1993); Dillon (1993); Miller (1992); Van Brunt (1988); Vigne (1995); Kremer and Perricaudet (1995); Haddadada et al. (1995); and Yu et al. (1994).

Non-viral delivery methods for nucleic acids and/or proteins include electroporation, lipid transfection, microinjection, biological projectiles, particle gun acceleration, virions, liposomes, immunoliposomes, lipid nanoparticles (LNPs), polycationic or lipid:nucleic acid conjugates, artificial virions, and reagent-enhanced nucleic acid uptake. Nucleic acid delivery can also be achieved through bacteria or viruses (e.g., *Agrobacterium*, *Rhizobium* sp., NGR234, *Sinorhizoboium meliloti*, *Mesorhizobium loti*, tobacco mosaic virus, potato virus X, cauliflower mosaic virus, and cassava vein mosaic virus) to plant cells (see, for example, Chung et al., 2006). Acoustic perforation using systems such as the Sonitron 2000 (Rich-Mar) can also be used for nucleic acid delivery. Cationic lipid-mediated protein and/or nucleic acid delivery has also been considered as an in vivo, in vitro or extracorporeal delivery method (see Zuris et al. (2015); also see Coelho et al. (2013); Judge et al. (2006); and Basha et al. (2011)).

Non-viral vectors (such as transposon-based systems, such as the recombinant Sleeping Beauty transposon system or the recombinant PiggyBac transposon system) can also be delivered to target cells and used for transposition of the polynucleotide sequence of the composition molecule or the polynucleotide sequence encoding the composition molecule in the target cells.

Other exemplary nucleic acid delivery systems include those provided by Amaxa.RTM.Biosystems (Cologne, Germany), Maxcyte (Rockville, Maryland), BTX Molecular Delivery Systems (Hollston, Massachusetts), and Copernicus Therapeutics (see, for example, U.S. Patent No. 6,008,336). Lipid transfection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipid transfection reagents are commercially available (e.g., Transfectam.TM, Lipofectin.TM, and Lipofectamine.TM.RNAiMAX). Cationic and neutral lipids suitable for recognizing effective receptors for polynucleotides in lipid transfection include those disclosed in PCT International Publications WO/1991/017424 and WO/1991/016024. Delivery can be made to cells (ex vivo administration) or target tissues (in vivo administration).

The preparation of lipid:nucleic acid complexes (including targeted liposomes, such as immunolipid complexes) is well known to those skilled in the art (e.g., see Crystal, Science (1995); Blaese et al. (1995); Behr et al. (1994); Remy et al. (1994); Gao and Huang (1995); Ahmad and Allen (1992); U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Another delivery method involves packaging the nucleic acid to be delivered into an EnGeneIC delivery vector (EDV). These EDVs are then specifically delivered to the target tissue using a bispecific antibody, where one arm of the antibody is specific to the target tissue and the other arm is specific to the EDV. The antibody carries the EDV to the surface of the target cells, where it is then carried into the cells via endocytosis. Once inside the cell, the contents are released (see MacDiarmid et al., 2009).

RNA or DNA virus-based systems are used for virus-mediated nucleic acid delivery, leveraging highly evolved processes to target viruses to specific cells in vivo and transport viral loads to the cell nucleus. Viral vectors can be administered directly to patients (in vivo) or used to process cells in vitro and administer the modified cells to patients (ex vivo). Conventional virus-based systems for nucleic acid delivery include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia virus, and herpes simplex virus vectors used for gene transfer.

The targeting of retroviruses can be altered by incorporating exogenous envelope proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells, typically producing high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats (LTRs) with a packaging capacity of up to 6–10 kb of exogenous sequence. Minimal cis-acting LTRs are sufficient for vector replication and packaging, which can then be used to integrate therapeutic genes into target cells to provide permanent transgenic expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibberish leukemia virus (GaLV), simmon immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (e.g., see Buchschacher et al. (1992); Johann et al. (1992); Sommerfelt et al. (1990); Wilson et al. (1989); Miller et al. (1991); PCT International Publication No. WO/1994/026877A1).

Currently, at least six viral vector methods are available for gene transfer in clinical trials. These methods utilize gene insertion into helper cell lines to complement defective vectors to produce transducers.

pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (see Dunbar et al., 1995; Kohn et al., 1995; Malech et al., 1997). PA317/pLASN was the first therapeutic vector used in gene therapy trials (Blaese et al., 1995). MFG-S packaging vectors have been observed to have transduction efficiencies of 50% or higher (Ellem et al., 1997; Dranoff et al., 1997).

Packaging cells are used to form viral particles capable of infecting host cells. These cells include 293 cells (which package adenoviruses, AAVs) and Psi-2 or PA317 cells (which package retroviruses). Viral vectors used in gene therapy are typically produced by production cell lines that package nucleic acid vectors into viral particles. The vectors usually contain the minimum viral sequence (if applicable) required for packaging and subsequent integration into the host; other viral sequences are replaced by expression cassettes encoding the proteins to be expressed. The missing viral functions are, in turn, provided by the packaging cell lines. For example, AAV vectors used in gene therapy typically only have the inverted terminal repeat (ITR) sequence of the AAV genome required for packaging and integration into the host genome. Viral DNA is packaged in a cell line containing helper plasmids encoding other AAV genes (i.e., rep and cap) but lacking the ITR sequence. This cell line is also infected with adenovirus as a helper virus. The helper virus promotes the replication of the AAV vector and assists in the expression of AAV genes in the plasmid. Due to the lack of the ITR sequence, the helper plasmid is not packaged in large quantities. Adenovirus contamination can be reduced, for example, through heat treatment, as adenoviruses are more sensitive to heat treatment than AAVs. Furthermore, AAVs can be produced on a clinical scale using baculovirus systems (see U.S. Patent No. 7,479,554).

In many gene therapy applications, it is desirable for gene therapy vectors to be delivered to specific tissue types with high specificity. Therefore, viral vectors can be modified to be specific to a given cell type by expressing ligands as fusion proteins with viral capsid proteins on the outer surface of the virus. Ligands with affinity for receptors known to be present on the target cell type are selected. For example, Han et al. (1995) reported that Moloney murine leukemia virus could be modified to express a human neural regulatory protein (heregulin) fused with gp70, and that the recombinant virus could infect certain human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs where the target cell expresses a receptor and the virus expresses a fusion protein containing a cell surface receptor ligand. For example, filamentous phages can be engineered to exhibit antibody fragments (e.g., FAB or Fv) with specific binding affinity to virtually any selected cell receptor. While the above description primarily applies to viral vectors, the same principle applies to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that favor uptake by specific target cells.

Gene therapy vectors can be delivered in vivo by administration to an individual patient, such as through systemic administration (e.g., intravitreal, intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or local administration.

Vectors can be delivered ex vivo to cells, such as cells transplanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, followed by re-implantation of the cells into the patient, optionally after selection of cells incorporating the vector. Non-limiting exemplary ex vivo methods may include retrieving tissue (e.g., peripheral blood, bone marrow, and spleen) from a patient for culture, transferring nucleic acids to the cultured cells (e.g., hematopoietic stem cells), and subsequently transplanting the cells into the patient's target tissue (e.g., bone marrow and spleen). In some embodiments, the stem cells or hematopoietic stem cells may be further treated with a viability enhancer.

In vitro cell transfection for diagnostic, research, or gene therapy purposes (e.g., by re-infusing transfected cells into a host organism) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from a test organism, transfected with a nucleic acid composition, and re-infused back into the test organism (e.g., a patient). Various cell types suitable for in vitro transfection are well known to those skilled in the art (e.g., see Freshney, *Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications* (6th edition, 2010), and the references cited therein regarding the discussion of how to isolate and culture cells from patients).

Vectors containing therapeutic nucleic acid compositions (e.g., retroviruses, liposomes, etc.) can also be directly applied to organisms for in vivo cell transduction. Administration can be carried out via any route typically used to introduce molecules into final contact with blood or tissue cells, including but not limited to injection, infusion, topical application (e.g., eye drops and ointments), and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and although more than one route can be used to administer a particular composition, a particular route often results in a more rapid and efficient response than another. According to some embodiments, the composition is delivered via intravenous injection.

Vectors suitable for introducing transgenes into immune cells (e.g., T cells) include non-integrating lentiviral vectors. See, for example, U.S. Publication No. 2009/0117617.

The pharmaceutically acceptable carrier depends in part on the specific composition being administered and the specific method of administration. Therefore, as described below, a variety of suitable pharmaceutical composition formulations are available for use (see, for example, Remington's Pharmaceutical Sciences, 17th edition, 1989).

The disclosed compositions and methods can also be used to manufacture medicines or immunotherapies for treating patients with cancer, disease, ailment, or infection.

Examples of RNA guide sequences that specifically target the SARM1 gene allele.

While numerous guide sequences can be designed to target the SARM1 gene, the nucleotide sequences described in Table 1, identified by SEQ ID NO: 1-13457, are specifically selected for effective implementation of the methods described herein. Examples of SARM1 biallelic knockout cells are provided in PCT International Application PCT/US2021/034583, the entire contents of which are incorporated herein by reference. According to some aspects of the invention, the guide molecules disclosed herein can be used to generate SARM1 biallelic knockout cells exhibiting increased viability and/or increased functionality compared to corresponding cells with wild-type or unmodified SARM1 expression. Non-limiting examples of cell types include liver cells (e.g., hepatocytes), lung cells, spleen cells, pancreatic cells, colon cells, skin cells, bladder cells, eye cells, ocular cells, retinal cells, corneal cells, brain cells, esophageal cells, head cells, neck cells, ovarian cells, testicular cells, prostate cells, placental cells, epithelial cells, endothelial cells, adipocytes, kidney/renal cells, heart cells, muscle cells, blood cells (e.g., white blood cells), immune cells, central nervous system (CNS) cells, ganglion cells, and combinations thereof.

Table 1 shows the guide sequences designed for association with the SARM1 allele as described in the above embodiments. Each engineered guide molecule is further designed to be associated with a target genomic DNA sequence located next to a prespacer adjacent motif (PAM), for example, a PAM matching the sequence NGG or NAG, where “N” is any nucleobase. The guide sequences are designed to work in conjunction with one or more different CRISPR nucleases, including but not limited to, for example, SpCas9WT (PAM SEQ: NGG), SpCas9.VQR.1 (PAM SEQ: NGAN), SpCas9.VQR.2 (PAM SEQ: NGNG), SpCas9.EQR (PAM SEQ: NGAG), SpCas9.VRER (PAM SEQ: NGCG), SaCas9WT (PAM SEQ: NNGRRT), SpRY (PAM SEQ: NRN or NYN), NmCas9WT (PAM SEQ: NNNNGATT), Cpf1 (PAM SEQ: TTTV), or JeCas9WT (PAM SEQ: NNNVRYM). The RNA molecules of this invention are each designed to bind to one or more different CRISPR nucleases to form a complex and are designed to target a specific polynucleotide sequence using one or more different PAM sequences corresponding to the CRISPR nuclease used.

Table 1: Guide sequence portions designed to be associated with specific SARM1 gene targets

The locations listed in column 1 of Table 1 are based on the gnomAD v3 database and UCSC Genome Browser assembly ID: hg38, sequencing/assembly provider ID: Genome Reference Consortium Human GRCh38.p12 (GCA_000001405.27). Assembly date: First released in December 2013; Patch 12 released in December 2017.

The embodiments provided below help to provide a more complete understanding of the present invention. The following embodiments illustrate exemplary modes of manufacturing and implementing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these embodiments, which are for illustrative purposes only.

Experimental details

Example 1: SARM1 Knockout Analysis

The guide sequence portion containing 17-50 consecutive nucleotides of high target activity was screened using SpCas9 in HeLa cells. This guide sequence portion comprised any one of the nucleotides shown in SEQ ID NO: 1-13457. Target activity was determined by DNA capillary electrophoresis.

Example 2: Additional SARM1 Edit Analysis

Sterile α- and Toll/interleukin-1 receptor motif 1 (SARM1) is an NAD+ hydrolase whose activity is correlated with axonal degeneration. To select the optimal RNA guide molecule for SARM1 biallelic knockout, 23 RNA guide molecules targeting SARM1 exons were screened in HeLa cells (Table 2). Briefly, SpCas9 encoding plasmids (64 ng) and DNA plasmids expressing RNA guide molecules (20 ng) were co-transfected in 96-well plates using a reagent (Polyplus). Cells were harvested 72 h after DNA transfection, genomic DNA was extracted, and capillary electrophoresis was performed using primers amplifying endogenous genomic regions. The plot in Figure 1A represents the mean of edit % ± standard deviation (STDV) from three (3) independent experiments. Capillary electrophoresis data analysis of all RNA guide molecules showed an activity range of 10% to 90%.

In addition, screening was performed in HeLa cells using OMNI-50 (SEQ ID NO: 13471) and OMNI-79 (SEQ ID NO: 13472) CRISPR nucleases. Transfection conditions were the same as those described for SpCas9 transfection. Editing efficiency was measured by next-generation sequencing (NGS) analysis. For OMNI-50, the g13 editing efficiency was 43% (STDV = 3.94). For OMNI-79, the g33 editing efficiency was 35% (STDV = 4.2). See Figure 1B. The guide sequence portions of the RNA guide molecules are listed in Table 4.

To verify that RNA guide molecules achieve Sarm1 knockout via nonsense-mediated decay (NMD), mouse Neuro-2a cells expressing SARM1 were used. To test the role of ten (10) most active guide RNA molecules targeting human SARM1 selected from HeLa, we identified ten (10) mouse-specific guide RNA molecules targeting mouse SARM1 DNA corresponding to human guide RNA molecules. The activity of mouse RNA guide molecules was tested in mouse cells. Briefly, 150 × 10³ Neuro-2a cells were mixed with a pre-assembled RNP consisting of 105 pmol of SpCas9 protein and 120 pmol of sgRNA (see Table 3), mixed with 100 pmol of electroporation enhancer (IDT-1075916), and electroporated using the SFCell4D-Nucleofector X Kit S (PBC2-00675, Lonza) with the DS-134 procedure. A subset of cells was harvested 72 hours after electroporation, and genomic DNA was extracted to measure the mid-target activity of NGS. According to NGS analysis, all guide RNA molecules showed high insertion or deletion (indel) activity (Figure 2).

To evaluate the effect of editing on SARM1 transcript levels, total RNA was extracted from Neuro-2a cells seven (7) days after electroporation, and Sarm1 mRNA levels were measured by qRT-PCR. The results showed that Sarm1 mRNA levels were reduced by more than 80% due to nonsense-mediated decay (NMD) (Figure 3).

Next, the editing of SARM1 by a novel CRISPR nuclease, OMNI-103 (SEQ ID NO: 13473), exhibiting unique PAM requirements, was tested. This nuclease was tested in HeLa cells as described above. For this purpose, OMNI-103 was transfected into HeLa cells using the corresponding OMNI-P2A-mCherry expression vector (pmOMNI, Table 7) and sgRNA molecules designed to target specific locations in the human genome (guide sequence portions (gRNA) sequences listed in Table 5A). At 72 hours, cells were harvested, and half of the cells were used by FACS to quantify transfection efficiency using mCherry fluorescence as a marker. The remaining cells were lysed, and their genomic DNA content was used for PCR reactions that amplified the corresponding putative genomic targets. The amplicones were subjected to NGS, and the percentage of editing events at each target site was calculated using the resulting sequences. Short insertions or deletions (indels) around the cleavage site are typical results of DNA end repair after nuclease-induced DNA cleavage. Therefore, the edit percentage is derived from the proportion of sequences containing insertions or deletions within each amplicon. See Table 5B and Figure 4.

Example 3: SARM1-edited NK cell assay

Metabolic test

Glycolysis and Mitochondrial Oxidative Metabolism: The Seahorse assay was performed according to the manufacturer's instructions, with modifications, to simultaneously analyze glycolysis and mitochondrial oxidative metabolism using the Seahorse XF Glycolysis Stress Assay Kit and the Seahorse XF Cellular Mitochondrial Stress Kit (Agilent Technologies). Briefly, NK and iNK cells were washed and resuspended in glucose-free medium (GIBCO). 1.5 × 10⁵ cells were seeded per well in triplicate and analyzed using a Seahorse Xfe96 analyzer (Agilent Technologies). Glucose, oligomycin, FCCP, sodium pyruvate, rotenone, and antimycin A were sequentially injected to measure metabolic function. SRC measurements were calculated as mean maximum OCR value minus mean basal OCR value. ATP-linked respiration was calculated as mean basal OCR value minus mean oligomycin value. Glycolysis was calculated as mean ECAR value after glucose minus mean basal ECAR value. Glycolytic reserve was calculated as mean maximum ECAR value minus mean ECAR value after glucose.

ATP quantification assay: 1 × 10⁵ NK and iNK cells per well were analyzed using the ATP bioluminescence assay kit HS II (Sigma Aldrich) and the results were analyzed using an Infinite M200 PRO spectrophotometer (Tecan).

NAD+ and NADH Quantification Assay: NAD+ and NADH concentrations were quantified using the NAD/NADH Cell Detection Kit (Cayman Chemical) according to the manufacturer's instructions and analyzed using an Infinite M200 PRO spectrophotometer. For oxidative stress analysis, NK and iNK cells were cultured with hydrogen peroxide (Sigma Aldrich) for 1 hour. After treatment, cells were cultured in serum-free medium containing 5 mM MitoSox indicator dye (Thermo Fisher). Cells were then washed, counterstained with anti-CD56 antibody and a viability-fixing dye for flow cytometry analysis. For mass spectrometry analysis of metabolites, iNK cells and expanded peripheral blood NK cells were rapidly frozen in liquid nitrogen and subjected to metabolomics analysis.

lethality test

In vitro assays for assessing cytotoxicity: Tumor target cells were stained with CFSE and resuspended in medium containing 5% FBS. NK effector cells and target cells were co-cultured in 96-well plates at a 5:1 E:T ratio in the presence of propidium iodide. The co-cultured cells were placed in an IncuCyte (Sartorius) live cell analysis system for up to 24 hours, and the cytotoxic ability of NK cells was quantified by double staining of cells (indicating target cell death).

3D Tumor Spheroid Cytotoxicity Assay: Tumor cells were cultured for several days to allow spheroid formation. Next, 4 × 10⁴ NK or iNK cells were gently added to each well, and the cells were co-cultured for several days. At the end of the culture, the cells in each well were lysed into a single-cell suspension and stained with a fluorescently conjugated CD56 antibody and a fixable viability dye for flow cytometry analysis. Tumor cells were quantified based on NucLight Red, and NK or iNK cells were quantified based on CD56 expression. In vivo preservation model of SARM1-edited NK cells:

The in vivo functionality of edited NK cells was tested in NSG mice using a retention model. Three experimental groups were used (control, edited cells, and unedited cells). Mice were irradiated with 300 rads the day before cell injection. Cells were resuspended in 200 μL of buffer and intravenously injected into mice. IL2 and IL15 were administered via intraperitoneal injection. Mice were sacrificed 3–4 days post-injection, and bone marrow and spleen were analyzed to determine NK cell retention capacity. A summary table of the experiment is shown below:

Table 2: Partial 20-nucleotide guide sequence targeting the human SARM1 coding sequence

Table 3: Partial 20-nucleotide guide sequence of the target mouse Sarm1 coding sequence

Table 4: Partial 22-nucleotide guide sequence of the target human SARM1 coding sequence

Table 5A: Partial 22-nucleotide guide sequence of the target sequence located in the SARM1 region

Table 5B: Quantitative results described in Figure 4

Table 6: Novel OMNI CRISPR nuclease, PAM requirements, and sgRNA scaffold sequence

Table 7: OMNI CRISPR nuclease mammalian expression plasmids and elements

Table 7 Appendix

Example 4: Knocking out SARM1 in NK cells can enhance the cytotoxic activity of NK cells.

Overview

Sterile α and Toll/interleukin-1 receptor motif 1 (SARM1) are NAD+ hydrolases, and their activity is related to axonal degeneration. GMX1778 is a potent and specific inhibitor of the NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). The selective inhibition of NAMPT by GMX1778 prevents NAD+ production and leads to cell death. Therefore, NAMPT is an important regulator of intracellular NAD concentration and thus a crucial regulator of energy metabolism. See Figure 5.

result

Editing and NGS: Two × 10⁶ thawed primary NK cells were grown for five (5) days and then electroporated with 113 pmol of SpCas9 and 226 pmol of SARM1_g91 gRNA (GUACUGGUGGCAAACCCAGU (SEQ ID NO: 11104)). After seven (7) days, the cells were sent for NGS analysis, which showed that the editing rate of SARM1 was 90%. See Figure 6A.

Kill assay: Ten (10) days after electroporation, 6250 NK cells were co-seeded with 2,500 GFP-labeled MCF7 target cells in triplicate. The co-cultures were incubated at an E:T ratio of 2.5:1 for 24 hours. Images were taken hourly. The kill assay medium was MEM-α supplemented with 10% FBS and 20 ng/ml IL-15. See Figure 6B.

Cell viability assay (ATPlite): Fifteen (15) days after electroporation, 50,000 SARM1_g91_KO and untreated (NT) cells were seeded in triplicate into gradually increasing concentrations of GMX1778 and incubated for 48 hours. Cell viability was quantified using the ATPlite assay kit. See Figure 6C.

in conclusion

These results indicate that SARM1 knockout NK cells exhibited increased cell viability after treatment with GMX1778 (a SARM1 inducer). Further results show that SARM1 knockout NK cells exhibited enhanced cytotoxicity.

Therefore, the results of this embodiment demonstrate that knocking out SARM1 in NK cells can enhance the cytotoxic activity of NK cells. Other combinations of nucleases and guide molecules can be used to knock out SARM1 and achieve the same effect. These unexpected results are the first to show that knocking out SARM1 in NK cells can enhance the cytotoxic activity of NK cells. Therefore, SARM1-KO NK cells can be used in immunotherapy, such as cancer treatment.

References

1.Ahmad and Allen(1992) "Antibody-mediated Specific Binging andCytotoxicity ofLipsome-entrapped Doxorubicin to Lung Cancer Cells in Vitro", Cancer Research 52:4817-20.

2.Anderson (1992) "Human gene therapy", Science 256:808-13.

3.Basha et al. (2011) "Influence of Cationic Lipid Composition on GeneSilencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells", Mol.Ther.19(12):2186-200.

4.Behr (1994) Gene transfer with synthetic cationic amphiphiles: Prospects for gene therapy”, Bioconjuage Chem 5:382-89.

5.Blaese(1995) "Vectors in cancer therapy:how will they deliver", Cancer Gene Ther.2:291-97.6.Blaese et al.(199 5) "T lympocyte-directed genetherapy for ADA-SCID: initial trial results after4years", Science 270(5235):475-80.

7.Buchschacher and Panganiban (1992) "Human immunodeficiency virusvectors for inducible expression of foreign genes", J.Virol.66:2731-39.

8. Burstein et al. (2017) "New CRISPR-Cas systems from uncultivatedmicrobes", Nature 542:237-41.

9.Chang and Wilson (1987) "Modification of DNA ends can decrease end-joining relative to homologous recombination in mammalian cells", Proc.Natl.Acad.Sci.USA 84:4959-4963.

10.Chung et al. (2006) "Agrobacterium is not alone:gene transfer toplants by viruses and other bacteria", Trends Plant Sci.11(1):1-4.

11.Coelho et al. (2013) "Safety and efficacy of RNAi therapy for transthyretin amyloidosis" N.Engl.J.Med.369,819-829.

12. Collias and Beisel (2021) "CRISPR technologies and the search for the PAM-free nuclease", Nature Communications 12,555.

13.Crystal(1995) "Transfer of genes to humans:early lessons andobstacles to success",Science270(5235):404-10.

14. Dillon (1993) "Regulation gene expression in gene therapy" Trends in Biotechnology 11(5):167-173.

15.Dranoff et al. (1997) "A phase I study of vaccination withautologous,irradiated melanoma cells engineered to secrete human granulocytemacrophage colon stimulating factor", Hum.Gene Ther.8(1):111-23.

16.Dunbar et al. (1995) "Retrovirally marked CD34-enriched peripheralblood and bone marrow cells contribute to long-term engraftment after autologous transplantation", Blood 85:3048-57.

17.Ellem et al. (1997) "A case report: immune responses and clinical course of the first human use of ganulocyte/macrophage-colony-stimulating-factor-tranduced autologous melanoma cells for immunotherapy", Cancer Immunol Immunother 44:10-20.

18.Gao and Huang (1995) "Cationic liposome-mediated gene transfer" GeneTher.2(10):710-22.19.Haddada et al. (1995) "Gene Therapy" "Using AdenovirusVectors", in: The Molecular Repertoire of Adenoviruses III: Biology and Pathogenesis, ed. Doerfler and pp. 297-306.

20.Han et al. (1995) "Ligand-directed retro-viral targeting of humanbreast cancer cells", Proc NatlAcad Sci U.S.A.92(21):9747-51.

21.Hu et al. (2018) "Evolved Cas9 variants with broad PAM compatibility and high DNA specificity", Nature 556(7699):57-63.

22. Jinek et al. (2012) "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Science 337(6096):816-21.

23.Johan et al. (1992) "GLVR1, a receptor for gibbon ape leukemia virus, is homologous to aphosphate permease of Neurospora crassa and is expressed athigh levels in the brain and thymus", JVirol 66(3):1635-40.

24.Judge et al. (2006) "Design of noninflammatory synthetic siRNAmediating potent gene silencing in vivo", Mol Ther.13(3):494-505.

25.Kohn et al. (1995) "Engraftment of gene-modified umbilical cordblood cells in neonates withadnosine deaminase deficiency", Nature Medicine 1:1017-23.

26. Kremer and Perricaudet (1995) "Adenovirus and adeno-associated virus mediated gene transfer", Br. Med. Bull. 51 (1): 31-44.

27. Macdiarmid et al. (2009) "Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug", NatBiotehcnol.27(7):643-51.

28. Malech et al. (1997) "Prolonged production of NADPH oxidase-corrected granulocyes after genetherapy of chronic granulomatous disease", PNAS 94(22):12133-38.

29.Miller et al. (1991) "Construction and properties of retroviruspackaging cells based on gibbonape leukemia virus", J Virol.65(5):2220-24.

30.Miller (1992) "Human gene therapy comes of age", Nature 357:455-60.

31. Mitani and Caskey (1993) "Delivering therapeutic genes–matching approach and application", Trends in Biotechnology 11(5):162-66.

32. Molday et al. (2013) "X-linked juvenile retinoschisis: Clinical diagnosis, genetic analysis, and molecular mechanisms", Prog Retin Eye Res. 31(3): 195-212.

33.Nabel and Felgner (1993) "Direct gene transfer for immunotherapy andimmunization", Trendsin Biotechnology 11(5):211-15.

34.Nishimasu et al. (2018) "Engineered CRISPR-Cas9 nuclease with expanded targeting space", Science 361(6408):1259-1262.

35.Remy et al. (1994) "Gene Transfer with a Series of Lipphilic DNA-Binding Molecules", Bioconjugate Chem.5(6):647-54.

36. Sentmanat et al. (2018) "A Survey of Validation Strategies for CRISPR-Cas9 Editing", ScientificReports 8:888, doi:10.1038/s41598-018-19441-8.

37.Sommerfelt et al. (1990) "Localization of the receptor gene for type D simian retroviruses onhuman chromosome 19", J.Virol.64(12):6214-20.

38.Van Brunt (1988) "Molecular framing: transgenic animals as bioactors" Biotechnology 6:1149-54.

39.Vigne et al. (1995) "Third-generation adenovectors for genetherapy", Restorative Neurologyand Neuroscience 8(1,2):35-36.

40.Walton et al. (2020) "Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants", Science 368(6488):290-296.

41.Weissman and Kariko (2015) "mRNA: Fulfilling the promise of genetherapy", MolecularTherapy(9):1416-7.

42.Wilson et al. (1989) "Formation of infectious hybrid virion withgibbon ape leukemia virus and human T-cell leukemia vi rus retroviral envelopeglycoproteins and the gag and pol proteins ofMoloney murine leukemia virus", J.Virol.63:2374-78.

43.Yu et al. (1994) "Progress toward gene therapy for HIV infection", Gene Ther.1(1):13-26.44.Zetsche e t al. (2015) "Cpf1 is a single RNA-guidedendonuclease of a class 2 CRIPSR-Cassystem" Cell 163(3):759-71.

45.Zuris et al. (2015) "Cationic lipid-mediated delivery of proteinsenables efficient protein basedgenome editing in vitro and in vivo" NatBiotechnol.33(1):73-80.

Claims (33)

1. A method of adoptive cell therapy or prevention comprising administering SARM1-inhibited or SARM1-inactivated cells to an individual suffering from cancer, infection, disease, or condition or identified as being at risk of cancer, infection, disease, or condition. 2. The method of claim 1, wherein SARM1-inhibited or SARM1-inactivated cells are modified to have reduced or inactivated SARM1 expression, or the cells are modified to express a dominant-negative SARM1 sequence variant or a dominant-negative fragment thereof. 3. The method according to claim 1 or 2, wherein the cell is selected from hematopoietic stem cells (HSC), induced pluripotent stem cells (iPS cells), iPSc-derived cells, natural killer cells (NK), iPS-derived NK cells (iNK), T cells, innate-like T cells (iT), natural killer T cells (NKT), γδ T cells, iPSc-derived T cells, inertial NKT cells (iNKT), iPSc-derived NKT, monocytes, or macrophages. 4. The method according to any one of claims 1-3, wherein, compared with corresponding cells having wild-type or unmodified SARM1 expression, SARM1-inhibited or SARM1-inactivated cells exhibit increased functionality, increased viability, increased persistence, increased proliferation, and/or increased tumor retention in an individual. 5. The method according to any one of claims 1-4, wherein SARM1-inhibited or SARM1-inactivated cells exhibit increased cytotoxicity and/or increased killing activity in an individual compared to corresponding cells having wild-type or unmodified SARM1 expression. 6. The method according to any one of claims 1-5, wherein the individual has cancer or is identified as being at risk of developing cancer. 7. The method of claim 6, wherein the cancer includes a tumor and/or the cancer is a hematologic malignancy. 8. The method according to claim 7, wherein the cancer is selected from static melanoma, metastatic prostate cancer, metastatic breast cancer, triple-negative breast cancer, bladder cancer, brain cancer, esophageal cancer, liver cancer, head and neck cancer, squamous cell lung cancer, non-small cell lung cancer, Merkel cell carcinoma, sarcoma, hepatocellular carcinoma, multiple myeloma, leukemia, non-Hodgkin lymphoma, lymphoma, B-cell lymphoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, cervical cancer, gastric cancer, kidney cancer, metastatic renal cell carcinoma, leukemia, ovarian cancer, and malignant glioma. 9. The method according to any one of claims 1-8, wherein the SARM1-inhibited or SARM1-inactivated cells are generated in vitro or ex vivo. 10. The method according to any one of claims 1-8, wherein the SARM1-inhibited or SARM1-inactivated cells are generated in vivo. 11. The method according to any one of claims 1-10, wherein the SARM1-inhibited or SARM1-inactivated cells are generated from cells obtained from an individual by mobilization and/or leukocyte separation. 12. The method of claim 11, wherein the cells are obtained from an individual via bone marrow aspiration. 13. The method according to any one of claims 1-12, wherein the cells are pre-stimulated prior to SARM1 inhibition or SARM1 inactivation. 14. The method according to any one of claims 1-13, wherein the SARM1-inhibited or SARM1-inactivated cells are cultured and expanded before being administered to an individual. 15. The method according to any one of claims 1-14, wherein SARM1-inhibited or SARM1-inactivated cells are capable of transplantation. 16. The method according to any one of claims 1-15, wherein SARM1-inhibited or SARM1-inactivated cells are capable of producing daughter cells. 17. The method of claim 16, wherein SARM1-inhibited or SARM1-inactivated cells are capable of producing progeny cells after transplantation. 18. The method of claim 17, wherein SARM1-inhibited or SARM1-inactivated cells are capable of generating progeny cells after autologous transplantation. 19. The method of claim 17 or 18, wherein SARM1-inhibited or SARM1-inactivated cells are capable of producing progeny cells for at least 12 months or at least 24 months after transplantation. 20. The method according to any one of claims 1-19, wherein SARM1-inhibited or SARM1-inactivated cells are generated by delivering gapmer, shRNA, siRNA, custom TALEN, macronuclease, zinc finger nuclease, CRISPR nuclease, or small molecule inhibitor to the cells. 21. The method according to any one of claims 1-19, wherein the alleles of the SARM1 gene in SARM1-suppressed or SARM1-inactivated cells undergo insertion or deletion mutations. 22. The method of claim 21, wherein an insertion or deletion mutation produces an early stop codon. 23. The method according to any one of claims 1-22, wherein SARM1-inhibited or SARM1-inactivated cells are generated by means comprising: Introducing a composition into cells, the composition comprising: At least one CRISPR nuclease, or a nucleotide molecule encoding a CRISPR nuclease; and An RNA molecule containing a guide sequence portion, or a nucleotide molecule encoding said RNA molecule. The complex of CRISPR nuclease and RNA molecules affects double-strand breaks in the SARM1 gene alleles. The guide sequence portion of the RNA molecule contains 17-50 consecutive nucleotides. 24. The method of claim 23, wherein the guide sequence portion is complementary to the target sequence located 50 base pairs upstream to 50 base pairs downstream of exon I, exon II, exon III, exon IV, exon V, exon VI, exon VII, exon VIII or exon IX of the SARM1 gene. 25. The method of claim 23 or 24, wherein the guide sequence portion is complementary to the target sequence located 30 base pairs upstream to 30 base pairs downstream of the exon of the SARM1 gene, and a) The exon is exon I, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 1-56, 301-356, 1348-2223, 57-114, 357-414, 2224-3095, 115-174, 415-474, 3096-3963 by no more than 3 nucleotides; b) The exon is exon II, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO:3964-7683 by no more than 3 nucleotides; c) The exon is exon III, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO:7684-8967 by no more than 3 nucleotides; d) The exon is exon IV, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO:8968-9525 by no more than 3 nucleotides; e) The exon is exon V, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO: 9526-10947 by no more than 3 nucleotides; f) The exon is exon VI, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO: 10948-11571 by no more than 3 nucleotides; g) The exon is exon VII, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO:11572-12717 by no more than 3 nucleotides; h) The exon is exon VIII, and the guide sequence portion contains a sequence that is identical to or differs from the sequence shown in any one of SEQ ID NO: 12718-13455 by no more than 3 nucleotides; or i) The exon is exon IX, and the guide sequence portion contains a sequence that is the same as or differs from the sequence shown in any one of SEQ ID NO:598-1347 by no more than 3 nucleotides. 26. The method according to any one of claims 23-25, wherein the guide sequence portion comprises 17-50 consecutive nucleotides, the 17-50 consecutive nucleotides containing nucleotides of any of the sequences shown in SEQ ID NO: 1-13457. 27. A drug comprising SARM1-inhibited or SARM1-inactivated cells, used for treating or preventing cancer, infection, disease, or ailment in an individual by any one of claims 1-26. 28. A kit for treating or preventing cancer, infection, disease, or condition in an individual, comprising the medicament of claim 27 and instructions for delivering the composition to an individual suffering from or identified as being at risk of cancer, infection, disease, or condition. 29. A method for inactivating the alleles of the sterile α- and toll/interleukin-1 receptor motif 1 (SARM1) gene in cells, the method comprising: Introducing a composition into cells, the composition comprising: At least one CRISPR nuclease, or a nucleotide molecule encoding a CRISPR nuclease; and An RNA molecule containing a guide sequence portion, or a nucleotide molecule encoding said RNA molecule. The complex of CRISPR nuclease and RNA molecules affects double-strand breaks in the SARM1 gene alleles. The guide sequence portion comprises 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain the nucleotides of the sequences shown in any one of SEQ ID NO: 1-13457, and The cells are selected from hematopoietic stem cells (HSC), induced pluripotent stem cells (iPS cells), iPSc-derived cells, natural killer cells (NK), iPS-derived NK cells (iNK), T cells, innate-like T cells (iT), natural killer T cells (NKT), γδT cells, iPSc-derived T cells, inertial NKT cells (iNKT), iPSc-derived NKT, monocytes, or macrophages. 30. A cell modified by the method of claim 29, wherein the modified cell contains at least one inactivated SARM1 allele. 31. The modified cells according to claim 30, used for adoptive cell therapy or prevention. 32. The modified cell of claim 31, wherein the adoptive cell therapy or prevention is for the treatment or prevention of an individual’s cancer, infection, disease, or condition. 33. A composition, method, process, kit, modified cell, or use, characterized by one or more elements disclosed herein.