Methods and Compositions for Inducing CAS Immunotolerance to Support CRISPR-CAS In Vivo Gene Editing

Abstract

Disclosed are tolerogenic compositions useful for inducing a tolerogenic immune response against a CRISPR-Cas effector polypeptide in a subject. The tolerogenic composition can comprise a) one or more microparticles, b) one or more regulatory T cell (Treg) stimulants encapsulated within each microparticle, and c) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. The tolerogenic composition can comprise a) a soluble microneedle array, b) one or more agents that promote the differentiation of tolerogenic DCs in the soluble microneedle array, and c) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 356,932, filed Jun. 29, 2022, which is incorporated herein by reference.

[0002] Description of Government Support This invention was made with government support under Grant No. AR074285 awarded by the National Institutes of Health (NIH) and Grant No. HR0011 - 17 - 2 - 0043 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.

[0003] Field of the Invention This application relates to the field of gene editing, and more specifically, to methods for inducing immune tolerance to CRISPR - Cas effector polypeptides.

[0004] Reference to Electronic Sequence Listing The contents of the electronic sequence listing named Sequences.xml, which is 12,288 bytes in size and has a creation date of Jun. 27, 2023, are incorporated herein by reference in their entirety.

Background Art

[0005] Background Gene editing is increasingly being used in therapeutic settings. To edit genes intracellularly in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas effector proteins, such as Cas9 and guide RNAs, are contacted with a target nucleic acid in a cell, and the CRISPR-Cas effector polypeptide and the guide RNA together modify the target nucleic acid or its expression. However, CRISPR-Cas effector polypeptides are foreign to mammalian hosts such as humans and can induce an immune response. Inducing antibodies and / or T cells specific for the CRISPR-Cas effector polypeptide can neutralize the CRISPR-Cas effector polypeptide and / or kill cells containing the CRISPR-Cas effector polypeptide. Such an immune response can reduce the efficiency of gene editing and prevent future editing using the CRISPR-Cas effector polypeptide. There is still a need for methods of inducing immune tolerance to CRISPR-Cas effector polypeptides, including but not limited to Cas9. Summary of the Invention Means for Solving the Problems

[0006] Summary of the Disclosure Disclosed are tolerogenic compositions useful for inducing a tolerogenic immune response against a CRISPR-Cas effector polypeptide in a subject. In some embodiments, the tolerogenic composition comprises a) microparticles, b) one or more regulatory T cell (Treg) stimulants encapsulated within the microparticles, and c) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. In other embodiments, the tolerogenic composition comprises a) a dissolving microneedle array, b) one or more agents that promote the differentiation of tolerogenic DCs in the dissolving microneedle array, and c) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof.

[0007] The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0020] Detailed description of some embodiments To facilitate in vivo editing without inducing an immune response, experiments were conducted to reduce the immune response to CRISPR-Cas effector polypeptides such as Cas9 and other editors. Reduction of the immune response, such as anti-Cas9 T cell or antibody responses, has at least two uses: i) improvement of the efficiency of the initial editing procedure, which often involves multiple edits with the same guide RNA, in the process of which an immune response to the editor is induced, resulting in death of cells that have undergone neutralization of the edit and / or the editor before reaching the cells in which the edit is to be made; improvement of the efficiency of the initial editing procedure; (ii) enabling the use of patients who have previously undergone an editing procedure to receive subsequent procedures for editing different genes. Tolerogenic compositions are disclosed herein for inducing a tolerogenic immune response to CRISPR-Cas effector polypeptides in a subject. In some embodiments, the tolerogenic composition comprises a) microparticles, b) one or more regulatory T cell (Treg) stimulants encapsulated within the microparticles, and c) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment. In other embodiments, the tolerogenic composition comprises a) a soluble microneedle array, b) a vitamin D analog, such as, but not limited to, MC903, and c) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. In some embodiments, the CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide, is co-administered with one or more additional agents.

[0021] The term Unless otherwise specified, technical terms are used according to their conventional usage. Definitions of common terms in molecular biology can be found in Krebs et al. (Eds.), Lewin’s Genes XII, published by Jones & Bartlett Publishers, 2017; and Meyers et al. (Eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published in 16 volumes by Wiley-VCH, 2008; and other similar references.

[0022] As used herein, the singular forms “a,” “an,” and “the” refer to both the singular and the plural unless the context clearly indicates otherwise. Further, “or” includes “and / or.” Thus, “a first particle or a second particle” includes “a first particle and / or a second particle,” and the compositions used in the methods herein can be used alone or in combination. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. The term “comprises” means “includes.” The term “about,” unless otherwise specified, means within five percent. Further, all base sizes or amino acid sizes, and all molecular weights or molecular mass values given for nucleic acids or polypeptides are approximate values provided for illustrative purposes.

[0023] Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0024] To facilitate the discussion of the various embodiments of the present disclosure, the following explanations of certain terms are provided.

[0025] Administration: Introduction of a composition, such as a small molecule inhibitor, to a subject by a selected route. Administration can be local or systemic. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0026] Agent: A drug, medicine, pharmaceutical, therapeutic agent, nutraceutical, biological molecule, or other compound that can be administered to a subject to effect a change, such as treatment, recovery, or prevention of a disease or disorder, or at least one symptom associated therewith, or a change in an immune response, including a regulatory immune response. An agent can generally be a "small molecule" having a molecular weight of about 2000 Daltons or less. An active agent can also be a "biological agent". Biological agents include proteins, antibodies, antibody fragments, peptides, oligonucleotides, and various derivatives of such materials.

[0027] Animal: A category that includes living multicellular vertebrate organisms, such as mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human subjects and veterinary subjects.

[0028] Cas9: An RNA-guided DNA endonuclease enzyme that can cut DNA. Cas9 has two active cleavage sites (HNH and RuvC), one on each strand of the double helix. Catalytically inactive (inactivated) Cas9 (dCas9) is also encompassed by the present disclosure. In some examples, dCas9 contains one or more of the following point mutations: D10A, H840A, and N863A.

[0029] The Cas9 nucleic acid and protein sequences are publicly available. For example, nucleotides 796693..800799 of GenBank® accession number CP012045.1 and nucleotides 1100046..1104152 of CP014139.1 disclose the Cas9 nucleic acid, and GENBANK® accession numbers AMA70685.1 and AKP81606.1 disclose the Cas9 protein. In some examples, Cas9 is an inactivated form of Cas9 (dCas9), such as those shown in GENBANK® accession numbers AKA60242.1 and KR011748.1 which are nuclease-deficient. In certain examples, Cas9 has at least 80% sequence identity, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to such sequences and retains the ability to cut DNA. Cas9 miniproteins are also included.

[0030] Caspase: An enzyme that is a cysteine-aspartic acid protease, cysteine aspartase, or cysteine-dependent aspartate-directed protease. Caspases are a family of protease enzymes that play an essential role in programmed cell death. They are called caspases due to their specific cysteine protease activity, and the cysteine in their active site nucleophilically attacks and cleaves target proteins only after an aspartic acid residue.

[0031] Conservative variant: A "conservative" amino acid substitution is a substitution that does not substantially affect or decrease the activity of a polypeptide. Specific non-limiting examples of conservative substitutions include the following examples.

Table 1

[0032] Co-administration: Administration of the agents disclosed herein with at least one other therapeutic or diagnostic agent within the same general period, and does not require administration at the exact same time (although co-administration includes administration at the exact same time). Thus, co-administration can be simultaneous or within a specified time frame. In certain embodiments, multiple therapeutic and / or diagnostic agents can be co-administered by encapsulating the agents within microparticles or by using the microneedles disclosed herein.

[0033] Control: A reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient or a subject treated with a carrier, non-target nucleic acid sequence, scrambled nucleic acid / amino acid sequence, or untreated cells from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient treated with an active agent. In still other embodiments, the control is a past control or a standard reference value or range of values (e.g., a previously tested control sample, e.g., a group of patients with a known prognosis or outcome, or a group of samples representing baseline or normal values).

[0034] Differences for immune responses, or differences in assays (e.g., ELISA, RNA expression profiles, etc.) performed on test samples and controls, can be an increase or conversely a decrease. The differences can be qualitative or quantitative differences, e.g., statistically significant differences. In some examples, the differences are an increase or decrease of at least about 5%, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or more than 500% compared to the control.

[0035] CRISPR (Clustered Regularly InterSpaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) editing system: An engineered nuclease system based on a bacterial system used for genome manipulation. This is based in part on the adaptive immune responses of many bacteria and archaea. Using such methods, genetic material can be added, removed, or altered at specific locations, e.g., target DNA or RNA sequences. Thus, the CRISPR / Cas system can be used for nucleic acid targeting (such as DNA or RNA) to, for example, detect target DNA or RNA, modify target DNA or RNA at any desired location, or cut target DNA or RNA at any desired location. Thus, such methods can be used to modify protein expression, for example, by introducing mutations into silene expression such as knocking out a gene.

[0036] In one example, the method edits DNA such as a genome and uses a “CRISPR-Cas effector polypeptide”. The CRISPR-Cas effector polypeptide is an enzyme that cleaves nucleic acids in this system. The CRISPR-Cas effector polypeptide includes, without limitation, Cas9. The CRISPR / Cas system can be engineered to cause DNA cleavage at a desired target within the genome of a cell and utilize the cell's endogenous machinery to repair the induced cleavage by homologous-directed repair (HDR) or non-homologous end joining (NHEJ). In another example, the CRISPR-Cas effector polypeptide cleaves RNA such as Cas13d nuclease (see, e.g., International Publication No. WO 2019 / 040664). The CRISPR-Cas effector polypeptide can be a type II, type V, or type IV CRISPR-Cas effector polypeptide. Non-limiting examples of CRISPR-Cas effector polypeptides include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas12, Cas10, Cas13d, Cpf1, C2c3, C2c2 and C2c1Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cas Lambda, CasX, CasY and Cas phi, as well as homologs thereof.

[0037] Dendritic cells (DC): Tolerogenic DCs are generally defined by including, in addition to increased antigen uptake capacity, low or medium levels of MHC II, co-stimulatory molecules CD80, CD86 and CD40, and chemokine receptor CCR7. Tolerogenic DCs can express high levels of inhibitory molecules such as Ig-like transcript (ILT) molecules (ILT3 / ILT4) and / or PD-L molecules (PD-L1, PD-L2). Furthermore, tolerogenic DCs can secrete small amounts of pro-inflammatory cytokines (IL-12p70) and large amounts of anti-inflammatory cytokines, such as IL-10. Tolerogenic DCs function to induce T cell anergy, T cell suppression, and the generation of regulatory T cells by several mechanisms, including the conversion of naive T cells to Tregs, the release of immunosuppressive cytokines, and the expression of functional indoleamine-2,3-dioxygenase (IDO).

[0038] Effective amount: The amount of an agent, such as an immunogen, that is sufficient to induce a desired response, such as a tolerogenic immune response, in a subject. It is understood that the procedures used to obtain a tolerogenic immune response to an antigen of interest may require multiple administrations of the disclosed compositions. Thus, an effective amount of the disclosed composition can be an amount of immunogen sufficient to induce a prime immune response in a subject, and the immunogen can then be boosted with the same or a different composition to induce a tolerogenic immune response.

[0039] Epitope: An antigenic determinant. These are specific chemical groups or peptide sequences on an antigenic molecule that elicit a specific immune response. For example, an epitope is the region of an antigen to which B cells and / or T cells respond. Epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of a protein.

[0040] Forkhead box P3 (Foxp3): A transcription factor that regulates and coordinates molecular processes involved in the differentiation and function of Tregs (Zheng and Rudensky, Nat. Immunol. 8:457-462, 2007). Treg cells are a type of T cell that play an important role in maintaining immune system homeostasis by suppressing hyperreactive immune responses (Josefowicz et al., Annu. Rev. Immunol. 30, 531-564, 2012). Defects in Treg cells can lead to autoimmune disorders and immunopathology. Conversely, certain tumors are enriched in Treg cells that suppress anti-tumor immune responses (Tanaka and Sakaguchi, Cell Res. 27, 109-118, 2017). An increase in Foxp3 activity enhances Treg suppressor function, while a decrease in Foxp3 activity suppresses Treg suppressor function (Loo et al., Immunity, 53, 143-157, 2020).

[0041] Immune response: The response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one embodiment, the response is specific to a particular antigen (“antigen-specific response”). In one embodiment, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, resulting in the production of specific antibodies. The immune response may be a “tolerogenic” immune response and thus may induce tolerance to a particular antigen. A tolerogenic immune response may include inducing and / or stimulating regulatory T cells (Tregs) and / or tolerogenic dendritic cells. Tolerogenic dendritic cells induce tolerance through several mechanisms. When stimulated, tolerogenic dendritic cells migrate to draining lymph nodes and present antigens to T cells via the interaction of MHC class II-antigen complexes on the dendritic cells and T cell receptors on the T cells. This can induce T cell clonal deletion, T cell anergy, or the proliferation of Tregs. Collectively, these mechanisms result in tolerance to a particular antigen. Thus, “immune tolerance” refers to a state of unresponsiveness of the immune system to substances, proteins, epitopes, or cells that would otherwise have the ability to induce an immune response in a given organism.

[0042] Isolated: An "isolated" biological component (e.g., a nucleic acid, protein, or cell) is substantially separated or purified from other biological components within the environment (such as a cell or tissue) in which the component exists, e.g., other chromosomes and extrachromosomal DNA and RNA, proteins, and cells. "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0043] Mammal: The term includes both human and non-human mammals. Similarly, the term "subject" includes both human subjects and veterinary subjects.

[0044] Microneedle: A microscopic structure associated with an "array," also referred to as a "microarray," that can penetrate the stratum corneum to facilitate transdermal or intradermal delivery of a therapeutic agent or sampling of fluid through the skin. "Array" refers to a medical device described herein that includes an ordered pattern of one or more structures that can pierce the stratum corneum to facilitate transdermal delivery of a therapeutic agent.

[0045] Microparticle: Microparticles generally refer to a general category that includes liposomes, nanoparticles, microspheres, nanospheres, microcapsules, nanorods, and nanocapsules. Microparticles may be composite structures and are not necessarily pure substances; they may be spherical or any other shape.

[0046] Optionally, the microparticles include one or more biodegradable polymers. As used herein, the term "biodegradable" refers to the ability of a material to be degraded by normal chemical, biochemical, and / or physical processes such as erosion, dissolution, corrosion, decomposition, hydrolysis, abrasion, and combinations thereof. Microparticles can have a diameter in the range of about 0.1 μm to about 1000 μm, or any range therebetween. Further information is provided below.

[0047] Adjust: To change in a statistically significant manner. The adjustment can be an increase or a decrease. One of ordinary skill in the art can identify appropriate assays for determining a statistically significant increase or decrease in a parameter. These include, but are not limited to, Student's t-test or paired t-test. Exemplary methods are provided in the Examples section.

[0048] Pharmaceutically acceptable carrier: Includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with pharmaceutical administration (see, e.g., Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition, 2005). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, balanced salt solutions, and 5% human serum albumin. Nonaqueous vehicles such as liposomes and fixed oils can also be used. Auxiliary active compounds can also be incorporated into the compositions. Practical methods for preparing administrable compositions include those provided in Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005).

[0049] Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). For polypeptides and proteins, the term "about" indicates an integer quantity. Thus, in one example, a polypeptide that is "about" 29 amino acids in length is 28 - 30 amino acids in length. Thus, a polypeptide of a particular number of residues "about" can be one amino acid shorter or one amino acid longer than the particular number. A fusion polypeptide contains the amino acid sequences of a first polypeptide and a second, different polypeptide (e.g., a heterologous polypeptide) and can be synthesized as a single amino acid sequence. A recombinant polypeptide has an amino acid sequence that is not naturally occurring or is made by the artificial combination of two other separated segments of an amino acid sequence.

[0050] Recombinant: A nucleic acid or protein having a sequence that does not occur naturally or having a sequence made by the artificial combination of two separated sequence segments (e.g., a "chimeric" sequence). This artificial combination can be achieved by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0051] Sequence identity: Similarity between amino acid sequences is expressed in terms of similarity between sequences and is otherwise called sequence identity. Sequence identity is frequently measured in terms of percent identity (or similarity or homology). The higher the percentage, the more similar the two sequences are. A homolog or variant of a polypeptide has a relatively high degree of sequence identity when aligned using standard methods.

[0052] Array alignment methods for comparison are known. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, 1970, J Mol Biol 48, 443-453; Higgins and Sharp, 1988, Gene 73, 237-244; Higgins and Sharp, 1989, CABIOS 5, 151-153; Corpet et al., 1988, Nucleic Acids Research 16, 10881-10890; Pearson and Lipman, 1988, Proc Natl Acad Sci USA 85, 2444-2448. Altschul et al., 1994, Nature Genet 6, 119-129 presents a detailed discussion of sequence alignment methods and homology calculations.

[0053] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990, J Mol Biol, 215, 403-410) is available from several information sources including the National Center for Biotechnology Information (NCBI, Bethesda, Maryland) and the Internet for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. An explanation of how to use this program to determine sequence identity is available on the NCBI web site on the Internet.

[0054] Homologs and variants of a polypeptide typically have at least 75%, for example at least 80% sequence identity, counted over the full-length alignment with the amino acid sequence of the polypeptide, using NCBI Blast 2.0, gapped blastp, set to default parameters. For comparisons of amino acid sequences over about 30 amino acids, the Blast 2 sequence function is used, with the default BLOSUM62 matrix set to default parameters (11 gap existence cost, 1 cost per residue gap). When aligning short peptides (less than about 30 amino acids), the alignment should be performed using the Blast 2 sequence function with the PAM30 matrix set to default parameters (penalty for open gap 9, extension gap 1). Proteins with even greater similarity to the reference sequence, when evaluated by this method, show an increase in the percentage of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is compared for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10 - 20 amino acids and can have at least 85% or at least 90% or 95% sequence identity depending on their similarity to the reference sequence. Methods for determining sequence identity in such short windows are available on the NCBI website on the Internet. Those skilled in the art will understand that these sequence identity ranges are provided for guidance only. It is entirely possible to obtain highly significant homologs outside the provided ranges.

[0055] Thus, in some instances, variants of a polypeptide or nucleic acid sequence typically have at least about 75%, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when counted over the full-length alignment with the amino acid or nucleotide sequence of interest. Sequences having even greater similarity to a reference sequence show an increase in percentage identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity when evaluated by this method. When less than the entire sequence is compared for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10-20 amino acids (or 30-60 nucleotides) and may have at least 85% or at least 90% or 95% sequence identity depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the NCBI website on the Internet.

[0056] As used herein, reference to "at least 90% identity" (or similar language) refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a particular reference sequence.

[0057] Single guide RNA (sgRNA): A synthetic guide RNA used to recognize a target DNA sequence and direct a Cas nuclease to the target. In some examples, the sgRNA is generated from subcloning of an optimized whole mouse genome lentiviral CRISPR sgRNA library such as lentiCRISPRv2-Brie (Doench et al., Nat Biotechnol 34:184-191, 2016, which is incorporated herein by reference in its entirety). In some examples, the sgRNA expression cassette further comprises a U6 promoter and / or a guide RNA scaffold.

[0058] Subject: A category that includes living multicellular vertebrate organisms, humans and non-human mammals, such as non-human primates, rats, mice, dogs, cats, horses, cows and pigs. In one example, the subject is a human. In a further example, a subject in need of modulating osteoclast fusion is selected. For example, the subject may be in need of an increase or decrease in osteoclast fusion, or an increase or decrease in bone resorption.

[0059] T cell: A white blood cell (lymphocyte) that is an important mediator of the immune response. T cells include, but are not limited to, cluster of differentiation (CD)4 + T cells and CD8 + T cells. Mature CD4+ cells, also known as helper T cells, help to orchestrate immune responses including antibody responses as well as killer T cell responses. Mature CD8 + T cells can be cytotoxic T cells. Activated T cells can be detected by increased cell proliferation and / or increased expression or secretion of one or more cytokines (e.g., IL-2, IL-4, IL-6, IFN-γ or TNFα). Activation of CD8 + T cells can also be detected by an increase in cytolytic activity in response to an antigen.

[0060] "Regulatory T (Treg) cells" are a type of T cell that plays a role in maintaining immune system homeostasis by suppressing hyperreactive immune responses (Josefowicz et al., Annu. Rev. Immunol. 30, 531-564, 2012). Tregs can be CD4+CD25+FoxP3+ T cells. Deficiency of Treg cells results in autoimmune disorders and immunopathology, although certain tumors are enriched in Treg cells that suppress the anti-tumor immune response (Tanaka and Sakaguchi, Cell Res. 27, 109-118, 2017). Tregs can also produce cytokines such as transforming growth factor (TGF)-β, interleukin (IL)-35, and IL-10. Other types of Tregs include Tr1 cells, which are CD4+FoxP3-IL10+TGFβ1+ cells (Gregori and Roncarolo, Front. Immunol., doi.org / 10.3389 / fimmu.2018.00233, February 15, 2018, incorporated herein by reference). Vitamin D3 induces the differentiation of both Treg cells and Tr1 cells (see van der Aar et al., J. Allerg. Clin. Immunol. 127(6):1532-1540.e7, 2011).

[0061] Summary In some embodiments, disclosed is a tolerogenic composition comprising: a1) one or more microparticles; b1) one or more regulatory T (Treg) cell stimulants encapsulated within each microparticle; and c1) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment.

[0062] In some embodiments, the one or more Treg stimulators include CCL22, retinoic acid, or vasoactive intestinal peptide. In further embodiments, the one or more Treg stimulators include IL-2, TGF-β, rapamycin, a rapamycin derivative, or a CCR8 ligand. In still other embodiments, the one or more Treg stimulators include TGFβ, IL2, and rapamycin. In further embodiments, the tolerogenic composition includes i) microparticles containing TGFβ, ii) microparticles containing IL-2, and iii) microparticles containing rapamycin.

[0063] In some embodiments, the microparticles include at least one polymer. In further non-limiting examples, the at least one polymer includes polyethylene glycol (PEG), poly(amino acid), polylactate, polylactic acid, polyglutamic acid, polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), poly(ethylene glycol-b-(DL-lactic-co-glycolic acid)-b-ethylene glycol) (PEG-PLGA-PEG), poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (PEG-b-PLGA), polycaprolactone-PEG (PCL-PEG), poly(vinylidene fluoride)-PEG (PVDF-PEG), poly(lactic acid-co-PEG) (PLA-PEG), poly(methyl methacrylate)-PEG (PMMA-PEG), and combinations thereof.

[0064] In other embodiments, the microparticles include alginate.

[0065] In still other embodiments, the microparticles have a diameter of about 0.5 to 5 μm and provide sustained release of TGF-β1, rapamycin, and / or IL-2. In certain non-limiting examples, the microparticles release for about one week.

[0066] In some embodiments, the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide. In other embodiments, the CRISPR-Cas effector fusion polypeptide comprises i) a CRISPR-Cas effector polypeptide, and ii) one or more heterologous effector polypeptides. In some embodiments, at least one of the one or more heterologous effector polypeptides is a single-stranded nuclease, a double-stranded nuclease, a helicase, a methylase, a demethylase, an acetylase, a deacetylase, a deaminase, an integrase, a recombinase, a base editor, or a prime editor.

[0067] In further embodiments, a method of inducing tolerance to a CRISPR-Cas effector polypeptide in a mammalian subject is disclosed, the method comprising administering to the subject an effective amount of the tolerogenic composition disclosed herein, thereby inducing tolerance to the CRISR-Cas effector polypeptide. In some embodiments, the composition is administered intradermally, subdermally, subcutaneously, or intramuscularly.

[0068] In other embodiments, the tolerogenic composition comprises a2) a soluble microneedle array, b2) one or more agents that promote the differentiation of tolerogenic DCs, and c2) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide.

[0069] In some embodiments, the soluble micron needle array comprises: i) a substrate comprising a biocompatible material forming a base portion; and ii) a plurality of micron needles extending from the base portion. In further embodiments, the biocompatible material comprises carboxymethylcellulose, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid (HA), or gelatin. In a specific non-limiting example, the biocompatible material comprises carboxymethylcellulose.

[0070] In further embodiments, the one or more agents that promote the differentiation of tolerogenic dendritic cells comprise vitamins or analogs thereof, such as vitamin D3 or vitamin D3 analogs.

[0071] Disclosed is a method of inducing tolerance to a CRISPR-Cas effector polypeptide in a mammalian subject, comprising administering to the subject an effective amount of a tolerogenic composition comprising a micron needle array. In further embodiments, the composition is administered locally, for example, intradermally or intracutaneously.

[0072] In still further embodiments, the tolerogenic composition is repeatedly administered to the subject. In a non-limiting example, the tolerogenic composition is administered to the subject in a prime-boost strategy.

[0073] In further embodiments, the disclosed method comprises administering, after administering the effective amount of the tolerogenic composition, a gene editing composition comprising a CRISPR-Cas effector polypeptide to the subject, wherein the tolerogenic composition induces tolerance to the CRISPR-Cas effector polypeptide present in the gene editing composition. In some embodiments, the gene editing composition is administered to the subject within about 6 months of administration of the tolerogenic composition. The subject can be a human. The subject can be a non-human mammal.

[0074] CRISPR-Cas effector polypeptide The disclosed method uses a tolerogenic composition comprising one or more CRISPR-Cas effector polypeptides or immunogenic fragments thereof, or one or more fusion polypeptides comprising a CRISPR-Cas effector polypeptide. Suitable CRISPR-Cas effector polypeptides for use are Class 2 CRISPR effector polypeptides, also referred to herein as Class 2 CRISPR-Cas effector polypeptides. For example, in some cases, the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide. In some cases, the type II CRISPR-Cas effector polypeptide is a Cas9 polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type V CRISPR-Cas effector polypeptide, such as a Cas12a, Cas12b, Cas12c, Cas12d or Cas12e polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type VI CRISPR-Cas effector polypeptide, such as a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide or a Cas13d polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas14 polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas14a polypeptide, a Cas14b polypeptide or a Cas14c polypeptide. Suitable CRISPR-Cas effector polypeptides for use include CRISPRi polypeptides. See, for example, Qi et al. (2013), Cell, 152:1173; and Jensen et al. (2021) Genome Research doi:10.1101 / gr.275607.121. Suitable CRISPR-Cas effector polypeptides for use include CRISPRa polypeptides. See, for example, Jensen et al. (2021) Genome Research doi:10.1101 / gr.275607.121; and Breinig et al. (2019) Nature Methods 16:51.Suitable CRISPR-Cas effector polypeptides include CRISPRoff polypeptides. See, for example, Nunez et al. (2021), Cell, 184:2503. Suitable CRISPR-Cas effector polypeptides include nickases. Suitable CRISPR-Cas effector polypeptides include catalytically inactive CRISPR-Cas effector polypeptides that retain binding to a target nucleic acid (when complexed with a guide RNA). Suitable CRISPR-Cas effector polypeptides include fusion polypeptides comprising i) a CRISPR-Cas effector polypeptide, and ii) one or more heterologous fusion partners (also referred to as "heterologous polypeptides"). Non-limiting examples of Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas13d, Cpf1, C2c3, C2c2 and C2c1Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cas lambda, Cas phi, Cas X, and Cas Y, and homologs thereof. Immunogenic fragments of these CRISPR-Cas effector polypeptides may also be included in the disclosed compositions.

[0075] In some cases, a CRISPR-Cas effector polypeptide suitable for inclusion in the compositions of the present disclosure is a Cas9 polypeptide. In some cases, the Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99% amino acid sequence identity with the Streptococcus pyogenes Cas9 amino acid sequence (SEQ ID NO: 1) shown in FIG. 11A.

[0076] In some cases, the Cas9 polypeptide is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, the saCas9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity with any known saCas9 amino acid sequence, such as the saCas9 amino acid sequence (SEQ ID NO: 2) shown in FIG. 11B.

[0077] In some cases, a suitable Cas9 polypeptide is a high-fidelity (HF) Cas9 polypeptide. Kleinstiver et al. (2016) Nature 529:490. For example, the amino acids N497, R661, Q695, and Q926 of the amino acid sequence shown in FIG. 1A are substituted, for example, with alanine. For example, the HF Cas9 polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence shown in FIG. 1A, and the amino acids N497, R661, Q695, and Q926 are substituted, for example, with alanine. In some cases, a suitable Cas9 polypeptide exhibits altered PAM specificity. See, for example, Kleinstiver et al. (2015) Nature 523:481.

[0078] In some cases, the appropriate Cas9 polypeptide comprises an R691A substitution. For example, in some cases, the appropriate Cas9 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 11A, and amino acid 691 is Ala.

[0079] In some cases, the appropriate Cas9 polypeptide comprises D1135V, R1335Q and T1337R substitutions. For example, in some cases, the appropriate Cas9 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 1A, amino acid 1135 is Val, amino acid 1335 is Gln, amino acid 1337 is Arg, and the Cas9 polypeptide exhibits relaxed PAM requirements.

[0080] In some cases, the appropriate Cas9 polypeptide is a SpRY variant. See, for example, Zhang and Zhang (2020) Trends Genetics 36:546; and Walton et al. (2020), Science, 368:290; and U.S. Patent Application Publication No. 2021 / 0284978. SpRY is a variant of S. pyogenes Cas9, and in this variant, the PAM requirement is reduced. For example, in some cases, the appropriate Cas9 polypeptide includes D1135L, S1136W, G1218K, E1219Q, R1335Q, and T1337R substitutions. For example, in some cases, the appropriate Cas9 polypeptide includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 11A, wherein amino acid 1135 is Leu, amino acid 1136 is Trp, amino acid 1218 is Lys, amino acid 1219 is Gln, amino acid 1335 is Gln, and amino acid 1337 is Arg. In some cases, the appropriate Cas9 polypeptide includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 11A. Includes amino acid substitutions at all 1, 2, 3, 4, 5, or 6 of the following positions: E1219 (e.g., E1219Q, E1219H, E1219S, or E1219V substitutions); S1136 (e.g., S1136W, S1136F, S1136A, or S1136V substitutions); D1135 (e.g., D1135L, D1135A, D1135W, or D1135F substitutions); G1218 (e.g., G1218R, G1218K, or G1218S substitutions); R1335 (e.g., R1335Q substitutions); and T1337 (e.g., T1337R or T1337K substitutions).

[0081] In some cases, the appropriate Cas9 polypeptide is a Cas9 polypeptide or a Cas9-NG polypeptide. See, for example, Zhong et al. (2019) Mol. Plant 12:1027; Hu et al. (2018), Nature, 556:57; Nishimasu et al. (2018) Science 361:1259. Cas9 nucleic acid and protein sequences are publicly available. For example, nucleotides 796693..800799 of GENBANK® accession number CP012045.1 and nucleotides 1100046..1104152 of CP014139.1 disclose Cas9 nucleic acid, and GENBANK® accession numbers AMA70685.1 and AKP81606.1 disclose Cas9 protein. In some examples, Cas9 is a deactivated form of Cas9 (dCas9), such as those shown in GENBANK® accession numbers AKA60242.1 and KR011748.1 which are nuclease-deficient. In certain examples, Cas9 has at least 80% sequence identity, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to such a sequence and retains the ability to cut DNA.

[0082] In some cases, the appropriate CRISPR-Cas effector polypeptide is a type V CRISPR-Cas effector polypeptide. In some cases, the type V CRISPR-Cas effector polypeptide is a Cas12a protein. In some cases, the Cas12a protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity to any known Cas12a protein, such as the Cas12a amino acid sequences shown in FIG. 11C or FIG. 11D.

[0083] In some cases, the CRISPR-Cas effector polypeptide is a CRISPR-Cas effector fusion polypeptide comprising a) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, and b) one or more heterologous polypeptides (also referred to as fusion partners). In some cases, the one or more heterologous polypeptides include a single-stranded nuclease, a double-stranded nuclease, a helicase, a methylase, a demethylase, an acetylase, a deacetylase, a deaminase, an integrase, a recombinase, a base editor, or a prime editor. In some cases, the one or more heterologous polypeptides include a nuclear localization signal. In some cases, the fusion partner (heterologous polypeptide) is a reverse transcriptase. In some cases, the fusion partner is a base editor. In some cases, the fusion partner (heterologous polypeptide) is a deaminase. The fusion polypeptide can also include a suitable carrier.

[0084] CRISPR-Cas effector polypeptides are known in the art and have reduced immunogenicity compared to parental CRISPR-Cas effector polypeptides (see PCT Publication No. WO 2017 / 081288A1, which is incorporated by reference). These recombinant CRISPR-Cas effector polypeptides include one or more amino acid substitutions at one or more residues corresponding to one or more MHC class I and / or MHC class II binding sites of the wild-type CRISPR-Cas effector polypeptide, and the recombinant CRISPR-Cas effector polypeptide has reduced immunogenicity compared to the wild-type CRISPR-Cas effector polypeptide.

[0085] Immunogenic fragments of CRISPR-Cas effector polypeptides, such as those corresponding to MHC class I and / or class II binding sites, are also useful. The immunogenic fragments can contain 9 (nonamer) or 10 (decamer) amino acids. The immunogenic fragments can induce a tolerogenic immune response. In further embodiments, the fragments are 8, 9, 10, 11, or 12 amino acids in length. Mixtures of immunogenic fragments are also useful. The mixtures can contain at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different fragments. These immunogenic fragments can be used to induce a tolerogenic immune response.

[0086] For the treatment of a subject, different doses may be required depending on the activity of one or more CRISPR-Cas effector polypeptides or their immunogenic fragments, or one or more fusion polypeptides, the mode of administration, age, and weight of the patient. In certain circumstances, higher or lower doses may be appropriate. Administration of the dose can be by single administration in the form of individual dose units or several smaller dose units, and by multiple administrations of fractionated doses at specific intervals. A skilled clinician can readily determine an effective dose for inducing tolerance.

[0087] For administration to a subject, an effective amount of one or more CRISPR-Cas effector polypeptides or immunogenic fragments thereof, or one or more fusion polypeptides comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, can be included in a pharmaceutically acceptable carrier. These pharmaceutical compositions can be prepared and administered in dosage units. Liquid formulations are generally useful. Solid dosage units are tablets, capsules, single-dose injections, and even suppositories. The appropriate administration format can be best determined by a physician for each individual subject. Various pharmaceutically acceptable carriers and their formulations are described in standard pharmaceutical treatises, such as Remington’s Pharmaceutical Sciences by E.W. Martin. See also Wang, Y.J. and Hanson, M.A., Journal of Parenteral Science and Technology, Technical Report No.10, Supp.42:2S, 1988. The dosage form of the pharmaceutical composition is determined by the selected mode of administration. Generally, the pharmaceutical composition comprises an effective amount of a CRISPR-Cas effector polypeptide, immunogenic fragment, and fusion polypeptide.

[0088] Suitable solid or liquid pharmaceutical dosage forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injection solutions in ampoule form, and formulations having sustained release of one or more CRISPR-Cas effector polypeptides or immunogenic fragments thereof, or one or more fusion polypeptides comprising a CRISPR-Cas effector polypeptide, and pharmaceutically acceptable excipients and additives and / or adjuvants, such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, solubilizers or scaffolds are customarily used. The pharmaceutical composition is suitable for use in various drug delivery systems. For a brief review of the methods for drug delivery, see Langer, Science 249:1527-1533, 1990.

[0089] The present disclosure is not limited to CRISPR-Cas effector polypeptides, immunogenic fragments, and fusion polypeptides. The compositions and methods of the present disclosure are used, among other things, with other systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).

[0090] Microparticles Provided are tolerogenic compositions comprising one or more microparticles, one or more Treg stimulants encapsulated within the one or more microparticles, and a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. Exemplary Treg stimulants include C-C chemokine motif (CCL22), interleukin 2 (IL-2), rapamycin, transforming growth factor beta (TGF-β), retinoic acid, and vasoactive intestinal peptide (VIP).

[0091] In certain embodiments, the Treg stimulant is highly effective in incrementing Tregs. In particular, CCL22, retinoic acid, and VIP increment Tregs. In certain embodiments, the therapeutic agent-bearing microparticles are highly effective in incrementing Tregs. The microparticles are effective in incrementing endogenous Treg cells to a local site.

[0092] In certain embodiments, the Treg stimulant is highly effective in inducing Tregs. In particular, IL-2, rapamycin, and TGF-β are effective in inducing Tregs. In certain embodiments, the therapeutic agent-bearing microparticles are highly effective in stimulating Tregs. Soluble microspheres are effective in stimulating endogenous Treg cells at a local site.

[0093] In certain embodiments, without being bound by theory, tolerance is naive CD4 +It is induced by the induction of the subject's own Tregs from T cells. This approach utilizes the body's natural mechanism to differentiate naive CD4 + T cells into Tregs through a subset of antigen-presenting cells known as tolerogenic dendritic cells (tDCs). Specifically, tDCs can induce the differentiation of Tregs through the secretion of IL-2 and TGF-β cytokines. However, the maintenance of Tregs is somewhat more complex and depends on a local microenvironment that is not only favorable for the differentiation of Tregs but also unfavorable for the differentiation into other effector T cells. One way to ensure that cells do not differentiate into pathogenic effector T cells is by small molecule rapamycin. Rapamycin (Rapa) is an mTOR inhibitor that can suppress the generation and proliferation of effector T cells. Rapamycin derivatives are also useful and are known in the art.

[0094] In certain embodiments, the body's endogenous Tregs are enriched by delivering a combination of Treg inducers through TRI microspheres (TGF-β1, rapamycin (Rapa) or its derivatives, and IL-2), which are microparticles encapsulating TGF-β1, Rapa, and IL-2. In some embodiments, TGF-β1, Rapa, and IL-2 are contained in separate microparticles and a mixture is administered. Thus, in some embodiments, the tolerogenic composition comprises i) microparticles containing TGFβ, ii) microparticles containing IL-2, and iii) microparticles containing rapamycin (or its derivatives). Without being bound by theory, this system can increase the prevalence of Tregs and thus induce tolerance.

[0095] One or more microparticles can be delivered by subcutaneous or intramuscular injection. The microparticles can be delivered to a mucosal surface such as a gel. One or more microparticles can be delivered by local injection or a local retention system. One or more microparticles are administered at or near the site of in vivo gene editing. One or more microparticles can be deleted at the same position / site as the CRISPR-Cas effector polypeptide, its immunogenic fragment, or the fusion polypeptide.

[0096] In certain embodiments, the amount of one or more Treg stimulants carried by one or more microparticles can range from about 1 ng to about 1 mg, more specifically from about 1 ng to about 100 μg of the agent per mg of microparticle. In certain specific embodiments, the amount of one or more Treg stimulants carried by one or more microparticles is about 25 ng to 7.5 μg of the agent per mg of microparticle. The Treg stimulant can be IL-2 or TGF-β1. The Treg stimulant can be included in separate microparticles or in one microparticle containing multiple Treg stimulants. As used herein, administration of "microparticles" is directed to the administration of one or more microparticles containing a Treg stimulant.

[0097] Suitable dosages include, but are not limited to, from about 5 μg to about 100 μg of IL-2 per about 200 mg of polymer. Suitable dosages include, but are not limited to, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μg of IL-2 per about 200 mg of polymer. Suitable dosages include, but are not limited to, from about 5 μg to about 100 μg of TGF-β1 per about 200 mg of polymer. Suitable dosages include, but are not limited to, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μg of TGF-β1 per about 200 mg of polymer. Suitable dosages include, but are not limited to, from about 1.5 mg to about 2 mg of rapamycin per about 200 mg of polymer. Suitable dosages also include, but are not limited to, from about 7.5 μg to about 2 mg of rapamycin per about 200 mg of polymer. Suitable dosages also include, but are not limited to, about 10 μg of rapamycin per 1 mg of polymer.

[0098] In yet other embodiments, the microparticles have a diameter of about 0.5 - 5 μm and provide sustained release of TGF-β1, rapamycin, and / or IL-2. In certain examples, the microparticles release for about one week.

[0099] The microparticles comprise at least one polymer. The polymer for the microparticles can be a biodegradable polymer as long as it is biocompatible. Biodegradable polymers include polyhydroxyacids such as polylactic acid and its copolymers. Exemplary polymers include polyglycolide, polylactic acid (PLA), and poly(lactic-co-glycolic acid) (PLGA). Another class of approved biodegradable polymers is polyhydroxyalkanoates. Other suitable polymers include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohol, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidone, polyglycolide, polysiloxanes, polyurethanes and their copolymers, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylic acid and methacrylic acid esters, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethylcellulose, cellulose triacetate, sodium salt of cellulose sulfate, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene polyethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohol), poly(vinyl acetate), polyvinyl chloride polystyrene, polyvinyl pyrrolidone, alginate, poly(caprolactone), dextran and chitosan. In some non-limiting examples, the microparticles can comprise alginate. In other non-limiting examples, the polymer is ester-terminated PLGA.

[0100] In a further embodiment, the polymer is a polyethylene glycol - poly(lactic acid - co - glycolic acid) copolymer. In further embodiments, at least one polymer is polyethylene glycol (PEG), poly(amino acid), polylactate, polylactic acid, polyglutamic acid, polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic acid - co - glycolic acid) (PLGA), polyvinyl acetate (PVA), poly(ethylene glycol - b - (DL - lactic acid - co - glycolic acid) - b - ethylene glycol) (PEG - PLGA - PEG), poly(ethylene glycol) - b - poly(D,L - lactide - co - glycolide) (PEG - b - PLGA), polycaprolactone - PEG (PCL - PEG), poly(vinylidene fluoride) - PEG (PVDF - PEG), poly(lactic acid - co - PEG) (PLA - PEG), poly(methyl methacrylate) - PEG (PMMA - PEG), and combinations thereof.

[0101] The loading percentage can be increased by "matching" according to the hydrophilicity or hydrophobicity of the polymer with respect to the drug to be encapsulated. In some cases such as PLGA, this can be achieved by selecting the monomer ratio such that the copolymer is more hydrophilic with respect to hydrophilic drugs or less hydrophilic with respect to hydrophobic drugs. Alternatively, the polymer can be made more hydrophilic, for example, by introducing carboxyl groups onto the polymer. In a non - limiting example, a combination of a hydrophilic drug and a hydrophobic drug can be encapsulated in microparticles prepared from a blend of a more hydrophilic PLGA and a hydrophobic polymer such as PLA.

[0102] In some embodiments, the polymer is a PLGA copolymer or a blend of PLGA and PLA. The molecular weight of PLGA is from about 10 kD to about 80 kD, more preferably from about 10 kD to about 35 kD. The molecular weight range of PLA is from about 20 to about 30 kDa. The ratio of lactide to glycolide is from about 75:25 to about 50:50. In one embodiment, the ratio is 50:50. Exemplary polymers include, but are not limited to, poly(D,L-lactic acid-co-glycolic acid) (PLGA, lactic acid to glycolic acid ratio 50:50, M n = 10 kDa, acid end, called 502H); poly(D,L-lactic acid-co-glycolic acid) (PLGA, lactic acid to glycolic acid ratio 50:50, M n = 25 kDa, acid end, called 503H); poly(D,L-lactic acid-co-glycolic acid) (PLGA, lactic acid to glycolic acid ratio 50:50, M n = 30 kDa, acid end, called 504H); poly(D,L-lactic acid-co-glycolic acid) (PLGA, lactic acid to glycolic acid ratio 50:50, M n = 35 kDa, ester end, called 504); and poly(D,L-lactic acid-co-glycolic acid) (PLGA, 75:25 lactic acid to glycolic acid ratio, M n = 10 kDa, called 752).

[0103] The microparticles can have a diameter in the range of about 0.1 μm to about 1000 μm, or any range therebetween, such as 0.1 μm to 0.5 μm, 0.5 μm to 1.0 μm, about 1.0 μm to about 5.0 μm, about 5.0 μm to about 10.0 μm, about 10.0 μm to about 20.0 μm, about 20.0 μm to about 35.0 μm, about 35.0 μm to about 50.0 μm, about 50.0 μm to about 75.0 μm, about 75.0 μm to about 100.0 μm, about 100.0 μm to about 250.0 μm, about 250.0 μm to about 500.0 μm, about 500.0 μm to about 750 μm, or about 750.0 μm to about 1000.0 μm. In one embodiment, the microparticles have a diameter in the range of about 0.1 μm to 1 μm, such as about 0.2 μm to about 1 μm, about 0.3 μm to about 1 μm, about 0.4 μm to about 1 μm, or about 0.5 μm to about 1 μm. In another embodiment, the microparticles have a diameter in the range of about 0.1 μm to 5 μm, such as about 0.2 μm to about 5 μm, about 0.3 μm to about 5 μm, about 0.4 μm to about 5 μm, or about 0.5 μm to about 5 μm. In certain embodiments, the supported microparticles can have a volume average diameter of 200 nm to 30 μm, more specifically 1 to 10 μm. In certain embodiments, the drug-loaded microparticles do not have a volume average diameter of 10 μm or more. The microparticles can be, for example, about 15 μm to 30 μm in diameter, such as about 15, 20, 25, or 30 μm in diameter. The microparticles can be, for example, about 0.5 μm to 5 μm in diameter, such as about 0.5, 1, 2, 3, 4, or 5 μm in diameter. The microparticles can be, for example, about 2 μm in diameter. The drug-loaded microparticles can contain few pores or, typically, various amounts of pores of various sizes controlled by adding NaCl during the synthesis process.

[0104] The method for preparing drug-loaded microparticles can be a single or double emulsion, depending on the solubility of the desired encapsulating agent in water, the degradation rate of the microparticles, and the molecular weight (MW can range from about 1000 Da to 100,000 Da) of the polymer chains used to prepare the microparticles that control the subsequent drug release kinetics.

[0105] The microparticles used in the methods disclosed herein can provide sustained release. For example, the sustained release can be over at least 1 day, more specifically at least 5 days or at least 10 days, and most specifically at least 30 days. The drug release can be linear or non-linear (single or multiple burst releases). In certain embodiments, the drug can be released without a burst effect. For example, the sustained release can exhibit a substantially linear release rate of the therapeutic agent in vivo over at least 1 day, more specifically at least 5 days or at least 10 days, and most specifically at least 30 days. Substantially linear release rate means that the therapeutic agent is released at a rate that varies by no more than about 20%, more typically no more than about 10%, over the desired period. It may be desirable to provide a relatively constant release rate of the drug from the delivery system over the lifetime of the system. For example, it may be desirable for the drug to be released in an amount of 0.1 to 100 μg per day, more specifically 1 to 10 μg per day, over the lifetime of the system. However, depending on the formulation of the polymeric microparticles, the release rate may increase or decrease.

[0106] In certain embodiments, the delivery system can release an amount of drug effective to provide a local concentration in the range of 1 pg / ml to 200 μg / ml, such as 1 to 5 μg / ml. Specific non-limiting embodiments are about 10 ng / ml of IL-2, about 5 ng / ml of TGF-β1, and about 10 ng / ml of rapamycin (see Jhunjhunwala et al., J. Controlled Release 159(1):78-84, 2012). In certain embodiments, there is no initial lag period of release. The desired release rate and target drug concentration can vary depending on the specific drug selected.

[0107] The microparticles used in the methods disclosed herein can provide sustained release of a drug. As used herein, the terms "sustained release" or "controlled release" refer to the leakage of any conjugated or encapsulated drug at a predetermined rate. For example, sustained release of a drug can occur from the predictable biodegradation of polymer particles (i.e., artificial antigen-presenting cells, for example). The biodegradation rate can be predetermined by altering the polymer composition and / or the ratio in which the particles are included. As a result, the sustained release can be short-term or the controlled release can be long-term.

[0108] In one embodiment, the short-term release is from 30 minutes to 1 hour. In one embodiment, the short-term release is from 1 hour to 3 hours. In one embodiment, the short-term release is from 3 hours to 10 hours. In one embodiment, the short-term release is from 10 hours to 24 hours.

[0109] In one embodiment, the long-term release is from 24 hours to 36 hours. In one embodiment, the long-term release is from 3 days to 7 days. In one embodiment, the long-term release is from 7 days to 1 month. In some embodiments, the microparticles provide sustained release of one or more Treg stimulators for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months. In some embodiments, the long-term release is about 1 week, for example, about 2 days to about 1 week, about 3 days to about 1 week, about 4 days to about 1 week, about 5 days to about 1 week, or about 6 days to about 1 week. The long-term release can be less than about 1 month.

[0110] In one embodiment, the long-term release is from 1 month to 6 months. In one embodiment, the long-term release is from 6 months to 1 year. In one embodiment, the long-term release is at least 1 month.

[0111] The tolerogenic composition containing the microparticles disclosed herein may include an excipient component, such as an effective amount of a buffering agent, and an antioxidant for protecting against the effects of ionizing radiation during sterilization. Suitable water-soluble buffering agents include, but are not limited to, alkali and alkaline earth carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates, etc., such as sodium phosphate, citrate, borate, acetate, bicarbonate, carbonate, etc. These agents are preferably present in an amount sufficient to maintain the pH of the system at about 2 to about 9, more preferably about 4 to about 8. Thus, the buffering agent can be about 5% (by weight) of the whole system. Suitable water-soluble preservatives include sodium bisulfite, sodium bisulfate, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, parabens, methylparaben, polyvinyl alcohol, benzyl alcohol, phenylethanol, etc., and mixtures thereof. These agents can be present in an amount of 0.001 to about 5% (by weight), preferably 0.01 to about 2% (by weight).

[0112] In certain embodiments, the microparticles disclosed herein can be administered by injection. The injection site includes, but is not limited to, intradermal, subcutaneous, hypodermic, or intramuscular administration. In some embodiments, the tolerogenic composition containing the microparticles is delivered by intravenous administration.

[0113] Microneedle array A CRISPR-Cas effector polypeptide, an immunogenic fragment thereof, or a fusion polypeptide can be administered with a soluble microneedle array. See, for example, U.S. Patent Application Publication No. US-2016-0271381-A1, which is incorporated herein by reference, and PCT Publication No. WO2020 / 232394, which is incorporated herein by reference. In some embodiments, the microneedle array is a tip-bearing microneedle array, which can be prepared using micro-milled master molds and spin molds. See U.S. Patent Application Publication No. US-2016-0271381-A1. As disclosed in PCT Publication No. WO2020 / 232394, undercuts or anchor features can improve skin retention during application and can be achieved without interfering with the processing steps, thereby allowing the MNA to be removed directly from the mold. Examples of the usefulness of microneedle devices include, for example, (1) co-delivery of the disclosed CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, with other agents for generating a tolerogenic immune response as needed, and (2) topical skin delivery.

[0114] Soluble microneedle arrays enable efficient and safe delivery to skin and mucosal surfaces. A fully soluble microneedle array substrate and unique microneedle geometry can be utilized to enable effective delivery of a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. This technology can also uniquely enable co-delivery of multiple chemically distinct agents for multifunctional drug delivery. The agents can promote the differentiation of tolerogenic dendritic cells, for example, by inducing a low responsiveness to partial sequence antigen exposure or by inducing T cells to differentiate into Tregs.

[0115] In some embodiments, the agent is vitamin D or an analog thereof, such as vitamin D3, or a vitamin D3 analog. Vitamin D3 has the following structure.

Chemical formula

[0116] Calcipotriene (also known as calcipotriol or MC-903) is a vitamin D3 analog known in the art. Calcipotriene has the following structure.

Chemical formula

[0117] Alfacalcidol may also be utilized. Alfacalcidol has the following structure.

Chemical formula

[0118] Tacalcitol may also be utilized. Tacalcitol has the following structure.

Chemical formula

[0119] Other suitable agents that promote the differentiation of tolerogenic DCs include, but are not limited to, retinoic acid, dexamethasone, IL-10, and TGF-β.

[0120] In some embodiments, provided herein is a transdermal insertion, e.g., a dissolving microneedle array for topical skin delivery, to a subject in need thereof for promoting tolerance to Cas9. The array includes a base portion, a plurality of microneedles extending from the base portion and containing a CRISPR-Cas effector polypeptide, an immunogenic fragment thereof, or a fusion polypeptide, and optionally an additional agent.

[0121] The microneedles can be pre-formed to have a shape including a first cross-sectional dimension at the top, a second cross-sectional dimension at the bottom, and a third cross-sectional dimension in the middle portion, where the middle portion is located between the top and the bottom, and the third cross-sectional dimension is larger than the first and second cross-sectional dimensions.

[0122] In yet other embodiments, each microneedle includes a plurality of layers of an insoluble biocompatible material. In some embodiments, manufacturing techniques are utilized that result in various active ingredients incorporated at the needle tip (see U.S. Patent Application Publication No. US-2016-0271381-A1, which is incorporated herein by reference). Thus, by localizing the active ingredient in this way, the remaining portion of the microneedle array volume contains a less expensive matrix material that is inactive and generally considered safe. The net result is (1) a reduction in the waste of undeliverable active ingredients incorporated in the non-needle portion of the microneedle array, and (2) a significant improvement in the efficiency of drug delivery based on a higher drug concentration at the skin-penetrating needle tip.

[0123] Thus, in some embodiments, the active ingredient is concentrated at the tip of the microneedles of each array. Thus, in contrast to conventional microneedle arrays, the support base structure has little or no active ingredient, so the active ingredient is not present at a uniform concentration across the microneedle array. Further, in some embodiments (e.g., as shown in FIGS. 3A, 3B, 4A, and 4B of U.S. Patent Application Publication No. US-2016-0271381, which is incorporated herein by reference), not only is there little or no active ingredient in the support structure, but the location of the active ingredient is concentrated in the upper half of the individual microneedles within the array. In some embodiments, the active ingredient is concentrated in the upper half of the individual microneedles. The active ingredient is concentrated at the tip of the microneedle, which is defined by the region of the microneedle that tapers and / or narrows extending from the base portion. The base portion extends from the support structure of the array.

[0124] As described above, in some embodiments, individual microneedles can contain the active ingredient only in the upper half of the microneedle. In other embodiments, individual microneedles can contain the active ingredient only at the tip of the microneedle or in a constriction near the tip. In still other embodiments, individual needles can contain the active ingredient throughout the entire microneedle portion extending from the support structure; see U.S. Patent Application Publication No. US-2016-0271381-A1, which is incorporated herein by reference.

[0125] The disclosed tolerogenic compositions can be delivered as disclosed in PCT Application No. PCT / US2016 / 057363, which is incorporated herein by reference. This PCT application discloses a microneedle array that can be configured to deliver their cargo (e.g., biologics or bioactive ingredients) through the stratum corneum to the epidermis and / or dermis while minimizing pain and bleeding by preventing penetration into deeper layers that may contain nerve endings and blood vessels. Pyramidal CMC microneedles can effectively penetrate the stratum corneum, epidermis, and dermis of living human skin and can thus be used for skin delivery. Thus, in some embodiments, the microneedle array comprises pyramidal CMC microneedles. In other embodiments, the microneedle array comprises obelisk-shaped needles.

[0126] To construct a microneedle array, a substrate can be used to form portions of each microneedle with and without a bioactive component. As described above, each microneedle can contain the bioactive component in only the microneedle, or in some embodiments, only the upper half of the microneedle, or in other embodiments, only a portion of the microneedle that tapers near the tip. Thus, in order to control the delivery of the bioactive component and the cost of the microneedle array, each microneedle can have a portion that contains the bioactive component (immunogen and / or adjuvant) and a portion that does not contain the bioactive component. In the embodiments described herein, the portion that does not contain the bioactive component includes the support structure of the microneedle array and, in some embodiments, the base portion (e.g., the lower half) of each microneedle within the array.

[0127] In some embodiments, a dissolving microneedle array includes a substrate that includes a biocompatible material forming a base portion, and a plurality of microneedles extending from the base portion. A variety of biocompatible materials can be used as the substrate for the microneedle array.

[0128] One bioactive component, such as a CRISPR-Cas effector polypeptide or a fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide and / or vitamin D or an analog thereof, can be covalently bound to the biocompatible material, such as by a disulfide bond. Optionally, the biocompatible material can be carboxymethyl cellulose. The bond can be cleavable in vivo by an enzyme and / or in response to pH, temperature, or both. One or more bioactive components can be the same or different bioactive components, and one or both of the bioactive components can be conjugated to the biocompatible material. The bioactive component can include, but is not limited to, a tolerogenic DC, a CRISPR-Cas effector polypeptide, an immunogenic fragment thereof, or an agent that promotes the differentiation of a fusion polypeptide.

[0129] The structural substrates of biodegradable solid microneedles most commonly include formulations based on poly(lactic-co-glycolic acid) (PLGA) or carboxymethylcellulose (CMC). However, other bases can also be used. Other polymeric materials include polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid (HA), gelatin, and / or mixtures of two or more of these. Other small molecule excipients, such as non-reducing sugars (e.g., trehalose, sucrose, etc.), may be included. PLGA is a biodegradable polymer that is not water-soluble, while CMC, PVP, PVA, HA, and gelatin are water-soluble.

[0130] In some embodiments, the biocompatible material is CMC. PLGA-based devices may limit drug delivery and protein delivery applications due to the relatively high temperatures (e.g., 135 degrees Celsius or higher) required for manufacturing, as well as vacuum and / or organic solvents. In contrast, CMC-based matrices can be formed at room temperature with simple spin-casting and drying processes, thereby making CMC microneedle arrays more desirable for the incorporation of CRISPR-Cas effector polypeptides or immunogenic fragments thereof, or fusion polypeptides comprising a CRISPR-Cas effector polypeptide or immunogenic fragment thereof.

[0131] CMC-hydrogel can be prepared from low-viscosity sodium salt of CMC with or without the active ingredients (described below) in sterile dH2O. In an exemplary embodiment, CMC can be mixed with sterile distilled water (dH2O) and the active ingredient to achieve a CMC concentration of about 25% by weight. The resulting mixture can be stirred until homogeneous and equilibrated at about 4°C for 24 hours. During this period, CMC and any other components can be hydrated to form a hydrogel. The hydrogel can be degassed in a vacuum for about one hour and centrifuged at about 20,000 g for one hour to remove residual micro-sized air bubbles that may interfere with the puncture / drying process of the CMC micro-needle array. The dry matter content of the hydrogel can be tested by drying a portion of it (10 g) at 85°C for about 72 hours. It is desirable to store the ready-to-use CMC-hydrogel at about 4°C until use.

[0132] Active ingredients such as CRISPR-Cas effector polypeptides, immunogenic fragments thereof, or fusion polypeptides containing CRISPR-Cas effector polypeptides or immunogenic fragments thereof, and optionally other agents such as vitamin D or vitamin D analogs, can be incorporated into the hydrogel of CMC at a relatively high (20 - 30%) CMC dry biopharmaceutical weight ratio prior to the spin-casting process. The array can be spin-cast at room temperature, and the process is compatible with the functional stability of a wide range of bioactive ingredients structurally. Since the master mold and production mold are reusable in multiple manufacturing cycles, the manufacturing cost can be significantly reduced. The resulting dehydrated CMC micro-needle array is generally stable at room temperature or slightly lower temperatures (such as about 4°C), maintains the activity of the incorporated biopharmaceutical, and facilitates easy and low-cost storage and distribution.

[0133] In an exemplary embodiment, the surface of the manufacturing mold can be covered with about 50 μl (for a mold with a diameter of 11 mm) of CMC-hydrogel and spin-cast by centrifugation at 2,500 g for about 5 minutes. After the first CMC-hydrogel layer, another 50 μl of CMC-hydrogel can be laminated onto the mold and centrifuged at 2,500 g for about 4 hours. At the end of the drying process, the CMC microneedle array can be separated from the mold, trimmed from excess material at the edges, collected, and stored at about 4 °C. The manufacturing mold can be washed and reused for further casting of the microneedle array.

[0134] In some embodiments, the CMC solid can be formed of a layer that does not contain an active ingredient and a layer that contains an active ingredient. Figures 11A-D of PCT application number PCT / US2016 / 057363, which is incorporated herein by reference, show CMC solids having different shapes (Figures 11A and 11B of PCT application number PCT / US2016 / 057363) and having active cargo embedded in an upper layer that becomes the portion of the microneedle having the active ingredient after micromilling. Figures 12A and 12B of PCT / US2016 / 057363 also show CMC solids having different shapes, Figure 12B of PCT / US2016 / 057363 shows a square, and Figure 12B shows a rectangle. Both CMC solids can be ground to dimensions for further processing described herein. It should be understood that the shapes are not intended to be limiting. Any shape can be used in the methods disclosed herein.

[0135] Generally, the microneedles have mechanical strength such that they remain intact for delivery while being inserted into the skin, for several days, and while being removed. The microneedles can have a straight or tapered shaft. In one embodiment, the diameter of the microneedle is largest at the proximal end of the microneedle and tapers from the proximal end to the distal end point. The microneedles can also be manufactured to have a shaft that includes both a straight (non-tapered) portion and a tapered portion.

[0136] The microneedle can be formed of a shaft having a circular cross-section vertically, or the cross-section can be non-circular. For example, the cross-section of the microneedle can be polygonal (e.g., star-shaped, square, triangular), rectangular, or another shape. The shaft can have one or more bores. The cross-sectional dimensions are typically about 10 nm to 1 mm, such as 1 μm to 200 μm, and more preferably 10 μm to 100 μm. The outer diameter is typically about 10 μm to about 100 μm, and the inner diameter is typically about 3 μm to about 80 μm.

[0137] The length of the microneedle is typically about 1 μm to 1 mm, such as about 1 μm to 50 μm, about 50 μm to 100 μm, about 100 μm to 250 μm, about 250 μm to 500 μm, about 500 μm to 750 μm, about 750 μm to 850 μm, or about 750 μm to 1 mm. The length is selected for a particular application considering both the insertion portion and the non-insertion portion. The array of microneedles can include, for example, a mixture of microneedles having various lengths, outer diameters, inner diameters, cross-sectional shapes, and spacing between the microneedles.

[0138] The microneedle can be oriented perpendicular or at an angle to the support structure of the microneedle array. Preferably, the microneedle is oriented perpendicular to the support structure, as a result, the density of the microneedles per unit area of the substrate can be increased. The array of microneedles can include a mixture of microneedle orientations, heights, or other parameters.

[0139] In some cases, the support structure of the microneedle array is typically about 50 mm 2 ~150 mm 2 、for example, about 100 mm 2 ~150 mm 2 、about 75 mm 2 ~100 mm 2 、about 50 mm 2 ~75 mm 2 、about 50 mm 2 ~65 mm 2、or about 60 mm 2 ~65 mm 2 and having an area of

[0140] In some cases, the substrate and / or the microneedles and other components are formed from a flexible material to allow the device to conform to the contours of a biological barrier such as the skin, blood vessel wall or eye to which the device is applied. The flexible device can facilitate more consistent penetration during use since penetration can be limited by bias of the attachment surface. For example, due to skin texture (i.e., small wrinkles) and hair, the surface of human skin is not flat.

[0141] In some cases, the tolerogenic composition comprising the microneedle array is administered intradermally. In some cases, the tolerogenic composition comprising the microneedle array is administered subdermally. In some cases, the tolerogenic composition comprising the microneedle array is administered subcutaneously.

[0142] Method for inducing tolerance The present disclosure provides a method for inducing tolerance to a CRISPR-Cas effector polypeptide in a mammalian subject, the method comprising administering to the subject an effective amount of the tolerogenic composition of the present disclosure. In some embodiments, the tolerogenic composition comprises a) one or more microparticles, b) one or more regulatory Treg stimulators encapsulated within each microparticle, and c) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. In other embodiments, the tolerogenic composition comprises a) a soluble microneedle array, b) a vitamin D analog or other agent, and c) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. The method generally comprises administering to a subject in need thereof an effective amount of the tolerogenic composition of the present disclosure.

[0143] In some cases, the methods of the present disclosure induce immune tolerance to a CRISPR-Cas polypeptide in a subject, thereby reducing the reactive immune response after administration of the CRISPR-Cas polypeptide to the subject. Accordingly, the present disclosure provides a method of reducing an immune response to a CRISPR-Cas polypeptide in a subject, the method comprising administering to the subject an effective amount of a tolerogenic composition.

[0144] Subjects suitable for treatment by the methods of the present disclosure include subjects in need of gene therapy or subjects to whom a gene editing composition is administered, such as for treating a disease or as an antiviral, anti-pathogen, or anti-cancer therapeutic agent, or for biological research. The subject can be a neonate, juvenile, or adult. Particularly interesting are mammalian subjects. Mammalian species that can be treated by the methods of the invention include canids and felids; horses; cows; sheep; etc. and primates, particularly humans. Animal models, particularly small mammals (e.g., mice, rats, guinea pigs, hamsters, lagomorphs (e.g., rabbits), etc.) can be used for experimental investigations. In some non-limiting examples, the subject is human. In other non-limiting examples, the subject is a veterinary subject including non-human primates. In some embodiments, the subject may have previously had a gene editing procedure with the same or different CRISPR-Cas effector polypeptides or immunogenic fragments thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. These subjects can be selected for treatment.

[0145] In some embodiments, an effective amount of the tolerogenic composition stimulates Tregs in the subject, such as by inducing or incrementally increasing Tregs, at one or more doses. Optionally, an effective tolerogenic composition increases the number of Tregs in the subject, at one or more doses. CD4 + , FOXP3 + and CD25 + Tregs and / or CD4 + FoxP3 - IL-10+ Tr1 cells can suppress reactive T cells. An effective tolerogenic composition can act on naive T cells at one or more doses and can include differentiation into Tregs. In some cases, an effective amount of a tolerogenic composition, when administered to a subject in need thereof at one or more doses, is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold or more than 2.5-fold increased in the number of Tregs in the subject as compared to the number of Tregs in the subject prior to treatment with the tolerogenic composition determined using the assays described herein or other assays known to those of skill in the art.

[0146] In some cases, an effective amount of a tolerogenic composition at one or more doses increases the number of tolerogenic DCs in a subject. In some cases, an effective amount of a tolerogenic composition, when administered to a subject in need thereof at one or more doses, is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold or more than 2.5-fold increased in the number of tolerogenic DCs in the subject as compared to the number of tolerogenic DCs in the subject prior to treatment with the tolerogenic composition.

[0147] In some cases, an effective amount of the tolerogenic composition in one or more doses increases the immune tolerance-inducing ability of tolerogenic DCs. The immune tolerance-inducing ability of tolerogenic DCs can be evaluated using techniques known to those skilled in the art. Using various assays known in the art, it can be evaluated whether the tolerogenic DCs described herein induce immune tolerance. In one aspect, the tolerogenic DCs described herein induce immune tolerance by creating an anti-inflammatory environment through an increase in the anti-inflammatory secretion of cytokines (e.g., IL-10) and a decrease in the secretion of pro-inflammatory cytokines (e.g., IL-12p70, IL-6, TNFα). In some cases, the ability of tolerogenic DCs to secrete IL-10, IL-12p70, IL-6, and TNFα is evaluated using an ELISA assay or a Luminex xMAP assay. In some cases, an effective amount of the tolerogenic composition in one or more doses increases the immune tolerance-inducing ability of tolerogenic DCs in a subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, or more than 2.5-fold compared to the immune tolerance-inducing ability of tolerogenic DCs in the subject prior to treatment with the tolerogenic composition.

[0148] In some embodiments, the tolerogenic composition, when administered to a subject in one or more doses, improves one or more effects related to the immunological response to a CRISPR-Cas effector polypeptide in the subject. In some examples, the tolerogenic composition reduces the number of CD4 + reactive T cells (i.e., the number of CD4 + T cells reactive with the CRISPR-Cas effector polypeptide), which in turn results in a decrease in CD8 + reactive with the CRISPR-Cas effector polypeptide. In some examples, the tolerogenic composition increases the number and / or activity of CD4 + Tregs, which in turn results in a decrease in CD4 + reactive T cells and / or CD8 +Reduce the number and / or activity of reactive T cells. These cells can be measured in a sample from a subject.

[0149] Methods for determining an immunological response to a CRISPR-Cas effector polypeptide are well known in the art. In some cases, the immunological response is determined by measuring the level of T cell activation induced by the CRISPR-Cas effector polypeptide. In some embodiments, the T cells are CD4 + and / or CD8 + T cells. In certain embodiments, the level of T cell activation is measured using methods well known in the art, including but not limited to flow cytometry, intracellular cytokine staining (ICS), staining of degranulation markers, and immunohistochemical staining. In certain embodiments, markers for T cell activation include, but are not limited to, CD137 and / or CD154 and / or CD107a (degranulation marker). In certain embodiments, activated T cells can be identified by the production of cytokines such as IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2). In some cases, the immunological response is measured by detecting the presence of Cas-specific antibodies by ELISA or other assays known in the art. The immunological response can also be evaluated by determining the number of Treg cells, such as by fluorescence-activated cell sorting for detecting the expression of CD4, CD25, and / or FoxP3.

[0150] In some embodiments, the tolerogenic composition of the present disclosure is administered to a subject before the gene editing composition comprising the CRISPR-Cas effector polypeptide is administered to the subject. The tolerogenic composition can be administered about 1 day to about 9 months before the gene editing composition, such as about 1 day to about 1 week, about 1 week to about 3 weeks, about 3 weeks to about 1 month, about 1 month to about 2 months, about 2 months to about 4 months, about 4 months to about 6 months, or about 6 months to about 9 months. In one embodiment, the gene editing composition is administered to the subject within about 6 months of the administration of the tolerogenic composition.

[0151] In some cases, the method includes administering to a subject a gene editing composition comprising a CRISPR-Cas effector polypeptide before administering the tolerogenic composition of the present disclosure. The gene editing composition can be administered 1 day to 9 months before the tolerogenic composition, for example, 1 day to 1 week before, 1 week to 3 weeks before, 3 weeks to 1 month before, 1 month to 2 months before, 2 months to 4 months before, 4 months to 6 months before, or 6 months to 9 months before the tolerogenic composition. In some cases, the gene editing composition is administered to the subject within 6 months of the administration of the tolerogenic composition. The gene editing composition can comprise the same CRISPR-Cas effector polypeptide or a different but related CRISPR-Cas effector polypeptide. In some embodiments, the subject has been previously administered a gene editing composition before administering the tolerogenic composition, and then, subsequently, another gene editing composition is administered after the administration of the tolerogenic composition. Thus, in this embodiment, the subject undergoes two or more gene editing procedures.

[0152] In some embodiments, the method includes administering to a subject a gene editing composition comprising a CRISPR-Cas effector polypeptide in addition to the tolerogenic composition of the present disclosure, wherein the tolerogenic composition induces tolerance to the CRISPR-Cas effector polypeptide present in the gene editing composition. The administrations can be simultaneous.

[0153] In these embodiments, the gene editing composition can include, but is not limited to, a guide RNA (gRNA) and a CRISPR-Cas effector polypeptide. Suitable CRISPR-Cas effector polypeptides for the gene editing composition include, but are not limited to, type II CRISPR-Cas polypeptides, type V CRISPR-Cas polypeptides, type VI CRISPR-Cas polypeptides, CRISPRi polypeptides, CRISPRa polypeptides, CRISPRoff polypeptides, and other CRISPR-Cas polypeptides optionally modified for the gene editing composition. In some cases, the CRISPR-Cas effector polypeptide suitable for inclusion in the gene editing composition includes a catalytically inactive CRISPR-Cas effector polypeptide that retains binding to the target nucleic acid (when complexed with the guide RNA). In some cases, the gene editing composition includes a gRNA or a nucleic acid encoding the guide RNA. In some cases, the gene editing composition includes a nucleic acid encoding a sequence to be inserted into the target nucleic acid.

[0154] The appropriate dosage of the disclosed tolerogenic composition can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical field, the dosage for any given patient depends on many factors including the patient's size, body surface area, age, the particular polypeptide or nucleic acid being administered, the patient's gender, time and route of administration, general health status, and other drugs being administered concurrently. The rate of repetition of administration can be determined based on the measured residence time and the concentration of the administered agent in body fluids or tissues. After successful treatment, it may be desirable to maintain the patient on maintenance therapy, in which case the tolerogenic composition of the present disclosure is administered at a maintenance dose for subsequent gene therapy procedures. Those skilled in the art will readily appreciate that the dosage levels can vary as a function of the type of tolerogenic composition, the route of administration, and the subject's sensitivity to side effects.

[0155] In some cases, multiple doses of the tolerogenic composition of the present disclosure are administered, such that the tolerogenic composition is repeatedly administered to the same subject. In some embodiments, at least two doses are administered as a prime and a boost. The prime and the boost can be the same dose or different doses.

[0156] The dosing frequency of the tolerogenic composition of the present disclosure can vary depending on any of a variety of factors, such as the severity of the symptoms, for example. For example, in some cases, the tolerogenic composition of the present disclosure is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), once every two weeks, once every three weeks, once every four weeks, twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid). When the tolerogenic composition of the present disclosure is administered intravenously, once a week, once every two weeks, once every three weeks, once every four weeks, or once a month dosing can generally be used at the start of treatment. The tolerogenic composition can be administered with the tolerogenic composition, administered to the subject in a prime-boost strategy, and complete tolerance is induced after the second (boost) administration.

[0157] The administration period of the tolerogenic composition of the present disclosure, for example, the period during which the tolerogenic composition of the present disclosure is administered, can vary depending on any of a variety of factors, such as the patient's response, for example. For example, the tolerogenic composition of the present disclosure can be administered over a period of about 1 day to about 1 week, about 2 weeks to about 4 weeks, about 1 month to about 2 months, about 2 months to about 4 months, about 4 months to about 6 months, about 6 months to about 8 months, about 8 months to about 1 year, about 1 year to about 2 years, or about 2 years to about 4 years, or beyond. The tolerogenic composition of the present disclosure is administered to the subject using any available method and route suitable for drug delivery, including in vivo and in vitro methods, as well as systemic and topical administration routes. Suitable administration methods are listed above.

[0158] Kit

[0159] Kits comprising tolerogenic compositions are provided herein. The tolerogenic compositions can comprise a1) microparticles, b1) one or more Treg stimulants encapsulated within the microparticles, and c1) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof. The kit can comprise multiple types of microparticles each encapsulating a different Treg stimulant, or one type of microparticle encapsulating more than two Treg stimulants.

[0160] The tolerogenic compositions can comprise a2) soluble microneedle arrays, b2) one or more agents that promote the differentiation of tolerogenic DCs, or c2) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof.

[0161] The kit can also include additional components to facilitate the particular use for which the kit is designed. For example, the kit can further comprise buffers and other reagents routinely used to perform a particular method. The kit can comprise a gene editing composition comprising a CRISPR-Cas effector polypeptide. The kit can include information that the tolerogenic composition induces tolerance to the CRISPR-Cas effector polypeptide present in the gene editing composition. The gene editing system can comprise a gRNA for the gene of interest.

[0162] The kit can comprise a container, a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from various materials such as glass or plastic. In some embodiments, the container can have an access port so that a specific amount of the agent can be withdrawn (e.g., the container can be a vial having a stopper pierceable by an intravenous solution bag or a hypodermic needle).

[0163] In some embodiments, the label or accompanying document indicates the use of a composition, for example, to induce tolerance. The accompanying document typically includes instructions that contain information regarding indications, dosages, contraindications, and / or warnings regarding the use of such a product, which are normally included in the commercial package of the product. The instructions for use may be written in electronic form (such as a computer disk or compact disk) or may be visual (such as a video file).

[0164] The present disclosure is illustrated by the following non-limiting examples.

Example

[0165] Gene editing using the CRISPR-Cas9 system has great potential for the treatment of various human diseases. The Cas9 nuclease used to delete or insert genes is derived from bacteria to which humans are commonly exposed (for example, S. pyogenes and S. aureus). Therefore, the majority of the population has existing humoral and cellular immunity against these Cas9 proteins, which is a potential barrier for safe and effective in vivo gene editing (see Charlesworth et al., Nature Medicine, 2019). In particular, Cas9-specific memory T cells can attack and kill the cells of patients in whom Cas9 has been delivered or in whom Cas9 expression has been induced. This can prevent the success of gene editing and may cause tissue or organ toxicity.

[0166] A method for measuring CD4 and CD8 T cell responses to Cas9 in mice was developed, enabling the evaluation of approaches to reduce immunogenicity. Cas9 peptides presented by MHC I and MHC II were identified in specific mouse strains. Furthermore, the method was used to induce immune tolerance (non-responsiveness) to Cas9 (see Figures 1A - 1C). Two drug delivery systems that teach the immune system to tolerate Cas9 and thereby potentially prevent harmful immune responses to Cas9 that interfere with in vivo gene editing. The first approach uses the injection of Cas9 protein along with biodegradable polymer microparticles that provide sustained delivery of regulatory T cell (Treg) inducing microparticles (TRI MPs), TGF-β1, rapamycin, and IL-2 to induce the differentiation of natural inhibitory Tregs. The second approach uses co-delivery of Cas9 protein in microneedles with, for example, the tolerogenic vitamin D3 analog (MC903). Delivery to the skin microenvironment was achieved by a soluble microneedle array (MNA). The disclosed approaches ultimately promote an increase in regulation of the inflammatory T cell ratio, thereby preventing or reducing harmful immune responses to cells that express or internalize Cas9 for the purposes of in vivo CRISPR gene therapy.

[0167] Example 1 Assays for measuring relevant Cas9 responses in mice To measure SpCas9-specific T cell immunity in C57BL / 6 mice, two robust immunization regimens and an ex vivo restimulation protocol were established. C57BL / 6 immunocompetent mice were immunized with SpCas9 protein and adjuvant to induce SpCas9-specific CD4 T cells, or injected with SpCas9-expressing cells to induce SpCas9-specific CD8 T cells. Similar methods can be used to study immunity to other editors.

[0168] To induce SpCas9-specific CD8 T cell immunity, splenocytes expressing SpCas9 intracellularly were isolated and activated overnight with 500 ng / mL lipopolysaccharide (LPS). These splenocytes were injected twice into naive C57BL / 6 mice (prime on day 0, boost on day 14). Splenocytes from immunized mice were restimulated in vitro with wild-type splenocytes or cells expressing Cas9. A statistically significant recall response against Cas9 was observed in animals immunized in the flank (Figure 2).

[0169] To elicit SpCas9-specific CD4 T cells, C57BL / 6 mice were immunized twice with SpCas9 protein formulated with TITERMAX™ Gold adjuvant (prime on day 0, boost on day 14). Splenocytes from immunized mice were harvested on day 21 and restimulated in vitro with SpCas9 protein. Only mice immunized with SpCas9 showed an antigen-specific recall response upon restimulation (Figure 3).

[0170] Example 2 Two different methods for inducing tolerance to Cas9 Results with tolerogenic microparticles. Cas9 co-delivered with microparticles (MP) containing rapamycin, TGF-β and IL-2 (Tri-MP) was injected subcutaneously into the nape of the neck of mice. The microparticles provide slow release of agents that induce the proliferation of specific Tregs and ultimately provide systemic tolerance to antigen-specific challenge. As shown in Figure 4B, proliferation of regulatory T cells was observed in the spleen after administration of Cas9 mixed with microparticles containing rapamycin, TGF-β and IL-2 ("Tri-MP Cas9"), but was not observed when rapamycin, TGF-β and IL-2 were excluded from the microparticles ("blank MP Cas9") or when Cas9 was excluded ("Tri-MP").

[0171] To investigate the effect on the immune response against Cas9, mice were pretreated with Tri-MP Cas9 and immunized 7 days later with Cas9 mixed with TITERMAX® adjuvant. Seven days later, mouse-derived splenocytes were tested by an Elispot assay for the presence of T cells that produce IFN-γ when stimulated with Cas9. Pretreatment with Tri-MP Cas9 almost completely suppressed the CD4 T cell response against Cas9 (Figure 4A). Surprisingly, a reduced response was also observed in mice pretreated with Tri-MP without Cas9, which may be due to the presence of residual immunosuppressive factors present at the time of Cas9 loading 1 week later. Therefore, the procedure was repeated and immunization was delayed until 14 days after MP administration. The response was boosted by a second immunization 1 week later, and 1 week after that, splenocytes were tested using a cytokine bead array to quantify IFN-γ produced in culture (Figure 4C). Pretreatment with TRI-MP mixed with Cas9 almost completely abrogated the T cell response. In contrast, pretreatment with TRI-MP alone or blank-MP mixed with Cas9 had no statistically significant effect. These results indicate that co-delivery of Cas9 and TRI-MP can expand Tregs and eliminate the Cas9 effector T cell response.

[0172] Results with tolerogenic microneedles: A second method of tolerance induction involves applying a tolerogenic microneedle array (MNA) containing Cas9 or the control protein BSA. Mice received three applications of the MNA. One week later, they were immunized subcutaneously at different sites with Cas9 protein formulated with TITERMAX® adjuvant. One week later, splenocytes were harvested from the mice and the T cell response against Cas9 was evaluated by intracellular cytokine staining. In particular, application of MNA with Cas9 reduced the response to subsequent stimulation (Figure 5). Surprisingly, the BSA control MNA was almost as effective as the Cas9 MNA. Although unexpected, the results suggest that application of MNA with Cas9 induces an immunosuppressive environment that reduces the subsequent immune response against Cas9.

[0173] Example 3 Figures 1A - 1C show the proposed mechanism by which TRIMP and MC903 MNA promote antigen - specific tolerance induction. For certain Cas9 applications, the Cas9 antigen can be administered subcutaneously with TRIMP rather than being administered to the skin near the TRIMP injection site.

[0174] TRIMP + Cas9 can be administered one or more times before in - vivo gene editing by the CRISPR - Cas9 system to reduce existing immunity to Cas9. Alternatively, TRIMP can be administered with the CRISPR - Cas9 in - vivo gene editing system (i.e., at the time of editing), depending on the administration site. Finally, TRIMP + Cas9 can be administered after the first gene editing and before one or more subsequent gene editings. Similarly, Cas9 + MC903 MNA can be administered one or more times before CRISPR - Cas9 gene editing and / or one or more times after the first round of gene editing and before subsequent rounds to reduce existing immunity to Cas9.

[0175] Mice were sensitized (immunized) to the S.pyogenes Cas9 (SpCas9) antigen by application of Cas9 (antigen) + poly(I:C) (adjuvant) MNA or by subcutaneous injection of Cas9 - expressing cells (specifically, splenocytes). Seven days later, the mice were challenged in the right ear via Cas9 MNA (re - exposed to the Cas9 antigen), and ear thickness was measured daily for the next 4 days. To control for increases in ear thickness not related to the Cas9 - antigen - specific inflammatory response, blank (empty) MNA was applied to the contralateral ear. Auricular swelling related to the delayed - type hypersensitivity (T - cell - mediated) response to Cas9 was reported as the change in ear thickness (delta) (i.e., Cas9 MNA - treated right ear - blank MNA - treated left ear). Greater auricular swelling is consistent with a stronger Cas9 - specific T - cell - mediated immune response (see Figure 6).

[0176] Before sensitization (immunization) with Cas9 + polyIC MNA, mice were treated by three consecutive applications of Cas9 + MC903 MNA (on days 0, 3, and 6). Three days later (day 9), mice were sensitized (immunized) to the Cas9 antigen by application of Cas9 + poly(I:C) MNA. Seven days later, mice were challenged in the right ear via Cas9 MNA (re-exposed to the Cas9 antigen), and ear thickness was measured daily for the next four days. To control for increases in ear thickness not related to the Cas9 antigen-specific inflammatory response, blank (empty) MNA was applied to the contralateral ear. Auricular swelling associated with the delayed-type hypersensitivity (T cell-mediated) response to Cas9 was reported as the change in ear thickness (delta) (i.e., Cas9 MNA-treated right ear - blank MNA-treated left ear). Greater auricular swelling is consistent with a stronger Cas9-specific T cell-mediated immune response and the pretreatment (preventive tolerization) with Cas9 + MC903 MNA preventing / reducing subsequent sensitization and the delayed-type hypersensitivity (auricular swelling) response. Unsensitized mice (gray) did not have an existing immunity to Cas9 prior to ear challenge with Cas9 MNA. The results are shown in Figure 7.

[0177] Four days after ear challenge (see Figure 7), auricular draining lymph nodes (DLNs) were isolated and the T cell response was evaluated by flow cytometry. In this model, Cas9 + polyIC MNA sensitization appears to mainly induce a Th1-mediated inflammatory-promoting effector T cell response, and pretreatment with Cas9 + MC903 MNA prior to sensitization significantly decreased the Th1 population and increased the Treg / Th1 and Treg / Teff ratios (see Figures 8A - 8C). This shift in the ratio of regulatory to effector T cells is consistent with the induction of a more tolerogenic immune response.

[0178] Unlike previous prophylactic tolerance induction models, tolerance was induced by treatment of previously sensitized mice (i.e., mice with existing immunity to Cas9) with Cas9+MC903 MNA. See FIGS. 9A-9B. In this therapeutic desensitization model, mice were first sensitized (immunized) to the S. pyogenes Cas9 (SpCas9) antigen by either application of Cas9+poly(I:C) MNA or subcutaneous injection of Cas9-expressing splenocytes. One week later, some of the mice were treated with Cas9+MC903 MNA (on days 7, 10, and 13). Five days after the last treatment with Cas9+MC903 MNA (day 18), all mice received adoptive transfer of a mixture of "target" cells (splenocytes) and "control" CFSE high cells (i.e., splenocytes that do not express Cas9). Since the target cells were labeled with the CFSE dye (10 uM) and the control cells were labeled with CFSE (1 uM), they could be identified and distinguished by flow cytometry. The next day, the spleens of the mice were processed and analyzed by flow cytometry to measure specific cell lysis (i.e., specific killing of Cas9-expressing target cells by Cas9-specific effector T cells, particularly cytotoxic T cells). The percent specific lysis was calculated as {1 - [(mean CFSE low / CFSE low ratio from naive mice) / (CFSE high / CFSE low ratio from immunized mice)]} × 100%. high

[0179] ​As expected, naive mice did not show specific lysis of Cas9-expressing target cells, consistent with the absence of Cas9-specific cytotoxic T cells. Mice primed with Cas9+ polyIC MNA also did not show specific lysis, suggesting that this priming method did not induce a robust cytotoxic T cell response. This is consistent with the results in Figures 8A-8C showing a dominant Th1 response and could result from the lack of cross-presentation of exogenous (extracellular protein) antigens to CD8+ T cells. In contrast, priming with subcutaneously injected Cas9-expressing splenocytes resulted in specific lysis of Cas9-expressing target cells (see Figures 9A-9B), and the degree of specific lysis was somewhat reduced by treatment with Cas9+ MC903 MNA after priming (see Figures 9A-9B). Since the prevention of lysis or death of Cas9-expressing cells by the immune system is the ultimate goal enabling in vivo editing by CRISPR-Cas9, this in vivo specific lysis assay is a relevant measure of the induction and suppression of tolerance of Cas9-specific T cell responses.

[0180] Pretreatment with SpCas9 and Mc903 MNA reduced priming and the subsequent delayed-type hypersensitivity response. C57BL / 6 mice were treated with MC903 MNA, SpCas9 MNA, or SpCas9+ MC903 MNA (days 0, 3, and 6) before being primed with SpCas9+ polyIC MNA (day 9). Control mice were primed but not tolerized (pretreatment). Five days after priming, the DTH response was induced by application of SpCas9 MNA to the right ear. Blank MNA was applied to the contralateral ear, and ear swelling 1-4 days after challenge was reported as the difference in ear thickness between the SpCas9 MNA-treated ear and the blank MNA-treated ear. Pretreatment with SpCas9+ MC903 MNA reduced the ear swelling DTH response to SpCas9 challenge (see Figure 12). In contrast, pretreatment with MC903 MNA or SpCas9 MNA had minimal effect on the ear swelling response, suggesting that co-delivery of SpCas9 and MC903 via MNA is required to reduce ear swelling (proof of reduction of antigen-specific T cell-mediated inflammation).

[0181] Figure 10 shows that sustained release of S. pyogenes (SpCas9) protein can be achieved by encapsulation into alginate hydrogel microparticles (MPs). Cas9 was loaded into low or medium viscosity alginate MPs crosslinked with CaCl2. In vitro release assays were performed by incubating a known mass of Cas9 MPs in PBS + 1% BSA buffer at 37 °C. At various time points, the suspension of MPs was centrifuged, the supernatant containing the released Cas9 was sampled, and the MPs were resuspended in fresh buffer. The concentration of Cas9 in the release buffer was measured by ELISA, and the cumulative release was calculated with respect to the amount of Cas9 released per mass of MPs. In Figure 10, the left graph shows the total cumulative release (ng Cas9 / mg MP), and the right shows the cumulative release as a percentage of the total encapsulated Cas9.

[0182] It will be apparent that the exact details of the described methods or compositions may be changed or modified without departing from the spirit of the described invention. The inventors claim all such modifications and variations that fall within the scope of the following claims and the gist thereof.

Claims

1. An immunogenic composition comprising: a1) one or more microparticles; b1) one or more regulatory T cell (Treg) stimulants encapsulated within the one or more microparticles; and c1) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof; or a2) a soluble microneedle array; b2) one or more agents that promote the differentiation of tolerogenic DCs in the soluble microneedle array; and c2) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof An immunogenic composition.

2. a1) the one or more microparticles; b1) the one or more regulatory T cell (Treg) stimulants encapsulated within the one or more microparticles; and c1) the CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising the CRISPR-Cas effector polypeptide An immunogenic composition according to claim 1, comprising.

3. The immunogenic composition according to claim 2, wherein the one or more Treg stimulants comprise CCL22, retinoic acid, or vasoactive intestinal peptide.

4. The immunogenic composition according to claim 2 or claim 3, wherein the one or more Treg stimulants comprise IL-2, TGF-β, rapamycin, a rapamycin derivative, or a CCR8 ligand.

5. The immunogenic composition according to any one of claims 2 to 4, wherein the one or more Treg stimulants comprise TGFβ, IL2, and rapamycin.

6. The immunogenic composition according to any one of claims 2 to 5, wherein the immunogenic composition comprises i) microparticles containing TGFβ, ii) microparticles containing IL-2, and iii) microparticles containing rapamycin.

7. The immunogenic composition according to any one of claims 2 to 6, wherein the one or more microparticles comprise at least one polymer.

8. The at least one polymer includes polyethylene glycol (PEG), poly(amino acid), polylactate, polylactic acid, polyglutamic acid, polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), poly(ethylene glycol-b-(DL-lactic-co-glycolic acid)-b-ethylene glycol) (PEG-PLGA-PEG), poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (PEG-b-PLGA), polycaprolactone-PEG (PCL-PEG), poly(vinylidene fluoride)-PEG (PVDF-PEG), poly(lactic-co-PEG) (PLA-PEG), poly(methyl methacrylate)-PEG (PMMA-PEG), and combinations thereof, the tolerogenic composition of claim 7.

9. The one or more microparticles include alginate, the tolerogenic composition according to any one of claims 2-6.

10. One or more microparticles are formulated for sustained release of the one or more regulatory T cell (Treg) stimulants, the tolerogenic composition according to any one of claims 1-9.

11. The CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide, the tolerogenic composition according to any one of claims 1-10.

12. The CRISPR-Cas effector fusion polypeptide includes i) a CRISPR-Cas effector polypeptide, and ii) one or more heterologous effector polypeptides, the tolerogenic composition according to any one of claims 1-11.

13. At least one of the one or more heterologous effector polypeptides is a single-stranded nuclease, a double-stranded nuclease, a helicase, a methylase, a demethylase, an acetylase, a deacetylase, a deaminase, an integrase, a recombinase, a base editor, or a prime editor, the tolerogenic composition of claim 12.

14. A method for inducing tolerance to a CRISPR-Cas effector polypeptide in a mammalian subject, said method comprising administering to said subject an effective amount of the tolerogenic composition according to any one of claims 2 to 13, whereby inducing tolerance to said CRISPR-Cas effector polypeptide.

15. The method according to claim 14, wherein said composition is administered intradermally, subdermally, subcutaneously or intramuscularly.

16. Said composition is a2) said soluble microneedle array, b2) said one or more agents that promote the differentiation of tolerogenic DCs in said soluble microneedle array, c2) a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or said immunogenic fragment The tolerogenic composition according to claim 1, comprising.

17. The tolerogenic composition according to claim 16, wherein said soluble microneedle array comprises i) a substrate comprising a biocompatible material forming a base portion, and ii) a plurality of microneedles extending from said base portion.

18. The tolerogenic composition according to claim 17, wherein said biocompatible material comprises carboxymethylcellulose, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid (HA), or gelatin.

19. The tolerogenic composition according to claim 18, wherein said biocompatible material comprises carboxymethylcellulose.

20. The tolerogenic composition according to any one of claims 16 to 18, wherein said one or more agents that promote the differentiation of tolerogenic DCs comprise vitamin D3 or a vitamin D3 analog.

21. A method for inducing tolerance to a CRISPR-Cas effector polypeptide in a mammalian subject, comprising administering to said subject an effective amount of the tolerogenic composition according to any one of claims 17 to 20, whereby inducing tolerance to said CRISPR-Cas effector polypeptide.

22. The method according to claim 21, wherein said composition is administered intradermally or subdermally.

23. The method according to any one of claims 14 to 15 or 21 to 22, wherein said tolerogenic composition is repeatedly administered to said subject.

24. The method according to claim 23, wherein the tolerogenic composition is administered to the subject in a prime-boost strategy.

25. The method according to any one of claims 14-15 or 21-24, comprising administering to the subject a gene editing composition comprising the CRISPR-Cas effector polypeptide after the step of administering the effective amount of the tolerogenic composition, wherein the tolerogenic composition induces tolerance to the CRISPR-Cas effector polypeptide present in the gene editing composition.

26. The method according to claim 25, wherein the gene editing composition is administered to the subject within about 6 months of the administration of the tolerogenic composition.

27. The method according to any one of claims 14-15 or 21-26, wherein the tolerogenic composition is administered to a subject previously administered a gene editing composition.

28. The method according to any one of claims 14-15 or 21-27, further comprising performing a gene editing procedure on the subject.

29. The method according to any one of claims 14-15 or 21-28, wherein the subject is a human.

30. The method according to any one of claims 14-15 or 21-28, wherein the subject is a non-human mammal.

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