Methods and compositions for CAS immune tolerance induction to support CRISPR-CAS in vivo gene editing

Abstract

A tolerogenic composition is disclosed that can be used to induce a tolerogenic immune response to a CRISPR-Cas effector polypeptide in a subject. In some embodiments, a tolerogenic composition comprises: a) one or more microparticles; b) one or more regulatory T cell (Treg) stimulators encapsulated within each of the microparticles; and c) a CRISPR-Cas effect polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effect polypeptide or an immunogenic fragment thereof. In other embodiments, a tolerogenic composition includes a) an array of dissolvable microneedles; b) one or more agents that promote differentiation of tolerogenic DCs in the dissolvable microneedle array; and c) a CRISPR-Cas effect polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effect polypeptide or an immunogenic fragment thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 356,932, filed on June 29, 2022, which is incorporated herein by reference. Government Support Statement

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

[0003] The present disclosure relates to the field of gene editing, and in particular to a method for inducing immune tolerance to CRISPR-Cas effector polypeptides. Electronic Sequence Listing Reference

[0004] The electronic sequence listing (named Sequences.xml) is 12,288 bytes in size and was created on June 27, 2023, the contents of which are incorporated herein by reference in their entirety. background

[0005] Gene editing is increasingly used in therapeutic environments. In order to edit genes in cells in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas effector proteins (e.g., Cas9) and guide RNAs are contacted with target nucleic acids in cells, wherein CRISPR-Cas effector polypeptides and guide RNAs together modify target nucleic acids or their expression. However, CRISPR-Cas effector polypeptides are foreign to mammalian hosts (e.g., humans) and may induce an immune response. Induced CRISPR-Cas effector polypeptide-specific antibodies and / or T cells can neutralize CRISPR-Cas effector polypeptides and / or kill cells including CRISPR-Cas effector polypeptides. This immune response can reduce the efficiency of gene editing and prevent future use of CRISPR-Cas effector polypeptides for editing. There is still a need to induce a method for immune tolerance to CRISPR-Cas effector polypeptides (e.g., but not limited to Cas9). SUMMARY OF THE DISCLOSURE

[0006] Disclosed are tolerogenic compositions that can be used to induce a tolerogenic immune response to 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) stimulators encapsulated within 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 soluble microneedle array; b) one or more agents that promote differentiation of tolerogenic DCs in a 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.

[0007] The foregoing and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A-Figure 1C .Schematic diagram. Figure 1A Antigens delivered alone or with adjuvants can promote innate inflammation, which triggers the migration of pro-inflammatory DCs to the skin draining lymph nodes (DLNs) and the differentiation of inflammatory effector T cells (Teff). Figure 1B TRI MPs delivered subcutaneously together with protein antigens (eg, Cas9) modulate the skin DLN microenvironment to promote regulatory T-cell (Treg) differentiation. Figure 1C Co-delivery of antigens (e.g., Cas9) with MC903 (vitamin D analog) microneedle arrays (MNAs) modulates the skin microenvironment and provides a tolerogenic environment for DCs to capture antigens, migrate to DLNs, and induce preferential Treg differentiation and / or reduce Teff differentiation.

[0009] Figure 2 Immunization with Cas9-expressing cells primes Cas9-specific CD8 + On days 0 and 14, 1×10 7 C57BL / 6 mice were immunized with LPS Cas9-Tg spleen cells. On day 26, spleen cells from immunized mice were harvested. Cells were incubated with control cells (B6) or cells expressing Cas9 antigen (Cas9) that were previously activated (+) or not activated (-) by LPS treatment. Intracellular cytokine staining is shown, which quantifies the production of IFNγ by CD8 +Frequency of T cells (unpaired t-test, *p<0.05, **p<0.01).

[0010] Figure 3 Immunization with adjuvanted Cas9 primes Cas9-specific CD4 + T cells. On days 0 and 14, C57BL / 6 mice were immunized with 100 μg SpCas9 or PBS buffer in Gold adjuvant. On day 21, splenocytes from mice immunized with PBS (left) or SpCas9 (right) were harvested and restimulated with Cas9 protein. CD4+ cells producing IFNγ assessed by intracellular cytokine staining are shown. + Frequency of T cells (two-way ANOVA, UN = unstimulated).

[0011] Figure 4A-4C : Treg-inducing microparticles with Cas9 expand splenic Tregs and reduce Cas9-specific CD4 T cell responses. C57BL / 6 mice were pretreated with PBS buffer, blank microparticles (MPs), or Treg-inducing microparticles (TRI-MPs) with or without Cas9 on day -12. Mice received one ( Figure 4A-4B ) or twice ( Figure 4C ) immunization. Mice were euthanized 7 days after the final immunization and IFNγ ELISPOT ( Figure 4A ), flow cytometry ( Figure 4B ) or cytokine bead array ( Figure 4C ) to evaluate spleen T cell responses. IFNγ responses elicited by stimulation with medium (open circles), OVA (diamonds), or Cas9 protein (squares) Figure 4A Statistical comparisons were performed using two-way ANOVA combined with Tukey's multiple comparison test. Figure 4B : Percentage of regulatory T cells in spleen. Ordinary one-way ANOVA, **p<0.01, ***p<0.001, ****p<0.0001. Figure 4C : Cytokine bead array quantification of IFNγ secretion after 3 days of stimulation, two-way ANOVA combined with Tukey's multiple comparison test.

[0012] Figure 5 : Tolerogenic microneedle arrays reduce Cas9-specific CD4 T cell responses. C57BL / 6 mice were left untreated (far left) or received intradermal application of Cas9 alone or MNAs containing Cas9 (50 μg) or BSA (50 μg) on ​​days 0, 3, and 6. Mice were immunized with Cas9 formulated with Gold at distal sites. Seven days after challenge, splenocytes were harvested and stimulated with medium (open circles) or Cas9 protein (filled squares). The frequency of IFNγ-secreting CD4 T cells obtained by intracellular cytokine staining is shown (two-way ANOVA with Tukey's multiple comparison test).

[0013] Figure 6 :SpCas9-specific delayed-type hypersensitivity (DTH) assay. Mice were sensitized (immunized) to Streptococcus pyogenes (S.pyogenes) Cas9 (SpCas9) antigens by applying Cas9 (antigen) + Poly (I: C) (adjuvant) MNA or by subcutaneous injection of splenocytes expressing Cas9. Seven days later, mice were challenged by Cas9 MNA at the right ear (exposed to Cas9 antigen again), and ear thickness was measured every day for the next four days. Blank (empty) MNA was applied to the contralateral ear to control any increase in ear thickness that was not related to the Cas9 antigen-specific inflammatory response. Ear swelling associated with T-cell-mediated DTH responses to Cas9 was reported as a change in ear thickness (Δ) (i.e., right ear treated with Cas9 MNA-left ear treated with blank MNA). Greater ear swelling was consistent with a stronger Cas9-specific T-cell-mediated immune response.

[0014] Figure 7 : Preventive tolerance is induced with Cas9 and tolerogenic vitamin D3 analog (MC903) microneedle arrays. Before sensitization with Cas9+PolyIC MNA, mice were treated by three consecutive applications of Cas9+MC903 MNA (day 0, day 3, and day 6). Three days later (day 9), mice were sensitized to the Cas9 antigen by applying Cas9+Poly (I:C) MNA. Seven days later, mice were attacked by Cas9 MNA at the right ear, and ear thickness was measured every day in the next four days. Blank MNA was applied to the contralateral ear to control any increase in ear thickness that was unrelated to the Cas9 antigen-specific inflammatory response. Ear swelling associated with T-cell-mediated DTH responses to Cas9 was reported as a change in ear thickness (Δ) (i.e., right ear treated with Cas9 MNA-left ear treated with blank MNA). Pretreatment with Cas9+MC903 MNA (preventive tolerance) reduced subsequent sensitization, resulting in less DTH (ear swelling) reaction.

[0015] Figure 8A-8C . Preventive tolerance induction using Cas9 and MC903 microneedle arrays. Four days after ear challenge (eg Figure 7 Auricular skin draining lymph nodes (DLN) were isolated and T-cell responses were evaluated by flow cytometry. Figure 8A-B : Total number of cells in each ear DLN: (1) CD4+FoxP3+ regulatory T cells (Treg), (2) CD4+T-bet+ type 1 helper T cells (Th1), (3) CD8+T-bet+ type 1 cytotoxic T cells (Tc1), (4) Treg / Th1 ratio, (5) Treg / (Th1+Tc1) or Treg (effector T cells, Teff) ratio. Figure 8C : The frequency of FoxP3+Treg, T-bet+Th1 and T-bet+Tc1 populations in ear DLN. The groups were compared by single factor independent ANOVA followed by Tukey's multiple comparison test. Significant differences (p<0.05, different letters) or non-significant differences (p>0.05, common letters) are indicated by concise letters. As a non-limiting example, the group labeled "a" is significantly different from the group labeled "b", but is not different from another group labeled a or the group labeled "ab". The group labeled "ab" is not significantly different from the group labeled "a" or the group labeled "b", but is significantly different from the group labeled "c".

[0016] Figure 9A-9B : Therapeutic desensitization with Cas9+Mc903 MNA. Tolerance was induced by treating previously sensitized mice (i.e., mice with pre-existing immunity to Cas9) with Cas9+MC903MNA. In the therapeutic desensitization model, mice were first sensitized to SpCas9 by subcutaneous injection of splenocytes expressing SpCas9. Starting one week later, some mice were treated with Cas9+MC903 MNA (days 7, 10, and 13). Five days after the last treatment with Cas9+MC903 MNA (day 18), all mice received CFSE expressing Cas9. 高 "Target" cells and "control" CFSE 低 The next day, the spleens of the mice were processed and analyzed by flow cytometry ( Fig. 9A ) to measure specific cell lysis (i.e., specific killing of target cells expressing Cas9 by Cas9-specific effector T cells (especially cytotoxic T cells)). The specific lysis percentage is calculated as ( Fig. 9B ). The sensitized and desensitized groups were compared by independent t-test.

[0017] Fig.10 : SpCas9 released from alginate hydrogel microparticles. Formulation: SpCas9 (2.5 mg) + 1% alginate (medium viscosity, 100 mg alginate) - marked as "b"; SpCas9 (2.5 mg) + 1% alginate (low viscosity, 100 mg alginate) - marked as "a".

[0018] Figures 11A-11D .Sequences of CRISPR-Cas effector polypeptides. SEQ ID NOs: 1-4 are shown.

[0019] Fig.12 Pretreatment with SpCas9+MC903 MNA reduces sensitization and subsequent delayed-type hypersensitivity (DTH) responses. Line graphs showing changes in ear thickness for each listed treatment are shown, as well as representative digital images of control animals and animals treated with SpCas9 and MC903 mRNA. Detailed description of several embodiments

[0020] Experiments were conducted to generate reduced immune responses to CRISPR-Cas effector polypeptides (e.g., Cas9 and other editors) to facilitate in vivo editing without inducing an immune response. The reduction of immune responses (e.g., anti-Cas9 T cell or antibody responses) has at least two applications: i) improving the efficiency of the initial editing procedure, which typically involves multiple rounds of editing using the same guide RNA, during which an immune response to the editor may be induced, resulting in the death of the cell being edited and / or being neutralized before the editor reaches the cell where editing is to occur; (ii) enabling patients who have previously undergone an editing procedure to undergo a subsequent procedure to edit a different gene. Disclosed herein are tolerogenic compositions that can be used to induce a tolerogenic immune response to 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) stimulators 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 includes a) a dissolvable 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. the term

[0021] Unless otherwise indicated, technical terms are used in accordance with conventional usage. Definitions of commonly used 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 by Wiley-VCH in 16 volumes, 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. In addition, "or" also includes "and / or"; therefore, "a first particle or a second particle" also 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, particularly suitable methods and materials are described below. The term "comprising" means "including". Unless otherwise indicated, the term "about" means within five percent. It should also be understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for descriptive purposes.

[0023] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, 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 the event of a conflict, this specification (including term explanations) shall prevail. In addition, materials, methods, and examples are exemplary only and are not intended to be limiting.

[0024] In order to facilitate review of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0025] Administration: Introducing a composition (e.g., a small molecule inhibitor) into 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, medicament, therapeutic agent, nutrient, biomolecule or other compound that can be administered to a subject to effect a change (e.g., to treat, ameliorate or prevent a disease or condition or at least one symptom associated therewith, or to alter an immune response, including a regulatory immune response). An agent can be a "small molecule," typically having a molecular weight of about 2000 Daltons or less. An active agent can also be a "biologic." Biologics include proteins, antibodies, antibody fragments, peptides, oligonucleotides, and various derivatives of such materials.

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

[0028] Cas9: RNA-guided DNA endonuclease that can cut DNA. Cas9 has two active cleavage sites (HNH and RuvC), corresponding to each strand of the double helix. The present disclosure also covers catalytically inactive (inactivated) Cas9 (dCas9). In some examples, dCas9 includes one or more of the following point mutations: D10A, H840A, and N863A.

[0029] Cas9 nucleic acid and protein sequences are publicly available. For example, Nucleotides 796693..800799 of accession number CP012045.1 and nucleotides 1100046..1104152 of CP014139.1 disclose Cas9 nucleic acid, and Accession numbers AMA70685.1 and AKP81606.1 disclose Cas9 proteins. In some examples, Cas9 is an inactivated form of Cas9 (dCas9), such as a nuclease-deficient form (e.g., In some 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. Also included are Cas9 mini-proteins.

[0030] Caspase: An enzyme that is a cysteine-aspartate protease, caspase, or cysteine-dependent aspartate-directed protease. Caspase is a family of proteases that play an important role in programmed cell death. It is named for its specific cysteine ​​protease activity, in which the cysteine ​​in its active site undergoes nucleophilic attack and cleaves the target protein only after the aspartate residue.

[0031] Conservative variants: "Conservative" amino acid substitutions are those that do not substantially affect or reduce the activity of a polypeptide. Specific non-limiting examples of conservative substitutions include the following: The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the polypeptide binds with the same affinity as the unsubstituted (parent) polypeptide. Non-conservative substitutions are substitutions that alter the activity of a polypeptide.

[0032] Co-administration: An agent disclosed herein is administered within the same general time period as at least one other therapeutic or diagnostic agent, and need not be administered at the same exact time point (although co-administration includes administration at the same exact time point). 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 using microneedles disclosed herein.

[0033] Control: Reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient or a subject treated with a vector, a non-targeted nucleic acid sequence, a 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 who has been treated with an active agent. In other embodiments, the control is a historical control or a standard reference value or range of values ​​(e.g., a previously tested control sample, such as a group of patients with a known prognosis or outcome, or a group of samples representing a baseline or normal value).

[0034] Difference, for example, for immune response or the difference in the determination (such as ELISA, RNA expression spectrum, etc.) carried out to test sample and control substance can be increase, or conversely be reduction.Difference can be qualitative difference or quantitative difference, such as statistically significant difference.In some instances, difference is to increase or decrease at least about 5% relative to control, such as 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%.

[0035] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated proteins) editing system: An engineered nuclease system based on a bacterial system for genome engineering. It is based in part on the adaptive immune response of many bacteria and archaea. Such methods can be used to add, remove, or change genetic material at a specific location (e.g., in a target DNA or RNA sequence). Therefore, the CRISPR / Cas system can be used for nucleic acid targeting (e.g., DNA or RNA), for example to detect target DNA or RNA, modify target DNA or RNA at any desired location, or cut target DNA or RNA at any desired location. Therefore, such methods can be used to modify the expression of proteins, such as silencing expression by introducing mutations, such as knocking out genes.

[0036] In one example, the method edits DNA, such as a genome, and uses a "CRISPR-Cas effector polypeptide". CRISPR-Cas effector polypeptides are enzymes that cleave nucleic acids in this system. CRISPR-Cas effector polypeptides include, but are not limited to, Cas9. The CRISPR / Cas system can be engineered to produce DNA breaks at desired targets in the genome of a cell, and utilize the cell's endogenous machinery to repair the induced breaks by homology-directed repair (HDR) or non-homologous end joining (NHEJ). In another example, the CRISPR-Cas effector polypeptide breaks RNA, such as the Cas13d nuclease (see, e.g., WO2019 / 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), Cas 12, 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, and their homologs.

[0037] Dendritic cells (DC): Tolerogenic DC are generally defined as including low or moderate levels of MHC II, co-stimulatory molecules CD80, CD86 and CD40, and chemokine receptor CCR7, in addition to having enhanced antigen uptake capacity. Tolerogenic DC 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). In addition, tolerogenic DC can secrete small amounts of pro-inflammatory cytokines (IL-12p70) and large amounts of anti-inflammatory cytokines (such as IL-10). Tolerogenic DC can secrete small amounts of pro-inflammatory cytokines (IL-12p70) and large amounts of anti-inflammatory cytokines (such as IL-10). Tolerogenic DC can secrete small amounts of pro-inflammatory cytokines (IL-12p70) and large amounts of anti-inflammatory cytokines (such as IL-10) through a variety of mechanisms, including the transfer of nascent antigens to T cells transform into Tregs, release immunosuppressive cytokines, and express functional indoleamine-2,3 dioxygenase (IDO) to induce T cell anergy, T cell suppression, and the generation of regulatory T cells.

[0038] Effective amount: An amount of an agent (e.g., an immunogen) sufficient to elicit a desired response, such as a tolerogenic immune response, in a subject. It should be understood that a procedure for obtaining a tolerogenic immune response against an antigen of interest may require multiple administrations of the disclosed compositions. Thus, an effective amount of a disclosed composition may be an amount of an immunogen sufficient to elicit a primary immune response in a subject, which may then be boosted with the same or a different composition to elicit a tolerogenic immune response.

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

[0040] Forkhead box P3 (Foxp3): a transcription factor that regulates and coordinates the molecular processes involved in Treg differentiation and function (Zheng and Rudensky, Nat. Immunol. 8: 457-462, 2007). Treg cells are a type of T cell that plays an important role in maintaining immune system homeostasis by suppressing overreactive immune responses (Josefowicz et al. Annu. Rev. Immunol. 30, 531–564, 2012). Treg cell defects can lead to autoimmune disorders and immunopathology. On the contrary, some tumors are rich in Treg cells that suppress anti-tumor immune responses (Tanaka and Sakaguchi, Cell Res. 27, 109–118, 2017). Increased Foxp3 activity enhances Treg suppressive function, while reduced Foxp3 activity inhibits Treg suppressive function (Loo et al., Immunity 53, 143–157, 2020).

[0041] Immune response: The response of an immune system cell (e.g., a B cell, a T cell, or a monocyte) to a stimulus. In one embodiment, the response is specific to a particular antigen (an "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 and results in the production of specific antibodies. The immune response may be a "tolerogenic" immune response, thereby inducing 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 a variety of mechanisms. Once stimulated, tolerogenic dendritic cells migrate to draining lymph nodes and present antigens to T cells through the interaction of MHC class II-antigen complexes on dendritic cells with T cell receptors on T cells. This can induce T cell clonal deletion, T cell anergy, or Treg proliferation. In summary, these mechanisms produce tolerance to specific antigens. Thus, "immune tolerance" refers to a state in which the immune system does not respond to substances, proteins, epitopes, or cells that are otherwise capable of eliciting an immune response in a particular organism.

[0042] Isolated: An "isolated" biological component (e.g., a nucleic acid, protein, or cell) has been substantially separated or purified from other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and cells) in the environment (e.g., a cell or tissue) in which the component is located. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

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

[0044] Microneedle: A microscopic structure related to an array, also called a "microarray", that is capable of piercing the stratum corneum to facilitate transdermal or intradermal delivery of a therapeutic agent or to collect a liquid sample through the skin. "Array" refers to a medical device described herein that includes an ordered pattern of one or more structures capable of piercing the stratum corneum to facilitate transdermal delivery of a therapeutic agent.

[0045] Microparticles: Microparticles generally refer to the general category that includes liposomes, nanoparticles, microspheres, nanospheres, microcapsules, nanorods and nanocapsules. A microparticle can be a composite structure and is not necessarily a pure substance; it can be spherical or any other shape.

[0046] In some cases, the microparticles include one or more biodegradable polymers. As used herein, the term "biodegradable" refers to the ability of a material to be broken down by normal chemical, biochemical, and / or physical processes (e.g., erosion, dissolution, corrosion, degradation, hydrolysis, abrasion, and combinations thereof). The diameter of the microparticles may range from about 0.1 μm to about 1000 μm, or any range therebetween. Additional information is provided below.

[0047] Modulate: change in a statistically significant manner. Modulation can be an increase or decrease. One skilled in the art can identify appropriate assays to determine a statistically significant increase or decrease in a parameter. These include, but are not limited to, Student's t-tests or paired ratio t-tests. The Examples section provides example methods.

[0048] Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like 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 stEdition, 2005). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextran solution, balanced salt solution, and 5% human serum albumin. Liposomes and non-aqueous solvents, such as fixed oils, may also be used. Supplementary active compounds may also be incorporated into the composition. Practical methods for preparing administrable compositions include 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 amino acid chain, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). For polypeptides and proteins, the word "about" refers to an integer number. Thus, in one example, a polypeptide that is "about" 29 amino acids in length is 28 to 30 amino acids in length. Thus, a polypeptide that is "about" a specified number of residues can be one amino acid shorter or one amino acid longer than the specified number. A fusion polypeptide includes the amino acid sequences of a first polypeptide and a second, different polypeptide (e.g., a heterologous polypeptide) and can be synthesized into a single amino acid sequence. A recombinant polypeptide has an amino acid sequence that is not naturally occurring or is composed of two originally separate amino acid sequence segments.

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

[0051] Sequence identity: Similarity between amino acid sequences is expressed as similarity between sequences, or also referred to as sequence identity. Sequence identity is often measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants of a polypeptide will have a relatively high degree of sequence identity when aligned using standard methods.

[0052] Methods for alignment of sequences 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; and Pearson and Lipman, 1988, Proc Natl Acad Sci USA 85, 2444-2448. Altschul et al., 1994, Nature Genet 6, 119-129, describes sequence alignment methods and homology calculations in detail.

[0053] NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990, J Mol Biol 215, 403-410) is available from a number of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to use this program to determine sequence identity is available on the NCBI website on the Internet.

[0054] Homologues and variants of polypeptides are generally characterized in that, using NCBI Blast 2.0, gap blastp is set to default parameters, counting in the full-length comparison with the amino acid sequence of polypeptide, there is at least 75%, for example at least 80% sequence identity. For the comparison of amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function is used, using the default BLOSUM62 matrix (gap existence cost is 11, and each residue gap cost is 1) set to default parameters. When comparing short peptides (less than about 30 amino acids), the Blast 2 sequence function should be used for comparison, using the PAM30 matrix (open gap 9, extension gap 1 penalty) set to default parameters. Albumins with even greater similarity to the reference sequence will show an increased identity percentage when evaluated by this method, for example 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, homologues and variants generally have at least 80% sequence identity within a short window of 10-20 amino acids, 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 within such a short window are available on the NCBI website on the Internet. Those skilled in the art will appreciate that these sequence identity ranges are provided for reference only; it is entirely possible to obtain strong and significant homologues beyond the provided ranges.

[0055] Therefore, in some examples, the variant of a polypeptide or nucleic acid sequence is generally characterized in that, when counted in the full-length comparison with the amino acid or nucleotide sequence of interest, there is at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Sequences with even greater similarity to the reference sequence will show an increased percentage identity when evaluated by the method, for example, 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, homologues and variants generally have at least 80% sequence identity in 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. The method for determining sequence identity in this short window is available on the NCBI website on the Internet.

[0056] As used herein, reference to "at least 90% identity" (or similar language) means "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 specified reference sequence.

[0057] Single guide RNA (sgRNA): a synthetic guide RNA for recognizing a target DNA sequence and guiding the Cas nuclease to the target. In some examples, sgRNA is generated by subcloning an optimized mouse whole genome lentiviral CRISPR sgRNA library (e.g., 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 living multicellular vertebrate organism, including the class of humans and non-human mammals, such as non-human primates, rats, mice, dogs, cats, horses, cattle, and pigs. In an example, the subject is a human. In another example, a subject is selected that needs to modulate osteoclast fusion. For example, the subject may need to increase or decrease osteoclast fusion, or may need to increase or decrease 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 (CD4) + T cells and CD8 + T cells. Mature CD4+ cells, also called helper T cells, help coordinate immune responses, including antibody responses as well as killer T cell responses. Mature CD8 + The T cells may be cytotoxic T cells. Activated T cells may be detected by increased cell proliferation and / or expression or secretion of one or more cytokines (e.g., IL-2, IL-4, IL-6, IFN-γ, or TNFα). CD8 + T cell activation can also be detected by an increase in cytolytic activity in response to antigen.

[0060] "Regulatory T (Treg) cells" are T cells that maintain the homeostasis of the immune system by suppressing excessive immune responses (Josefowicz et al. Annu. Rev. Immunol. 30, 531–564, 2012). Tregs can be CD4+CD25+FoxP3+T cells. Treg cell defects lead to autoimmune diseases and immunopathology, and some tumors are rich in Treg cells that suppress anti-tumor immune responses (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 Treg include Tr1 cells, which are CD4+FoxP3-IL10+TGFβ1+ cells, see Gregori and Roncarolo, Front.Immunol., doi.org / 10.3389 / fimmu.2018.00233, February 15, 2018, which is incorporated herein by reference. Vitamin D3 induces Treg and Tr1 cell differentiation, see van der Aar et al., J.Allerg.Clin.Immunol.127(6):1532-1540.e7, 2011. Overview

[0061] In some embodiments, a tolerogenic composition is disclosed, comprising a1) one or more microparticles; b1) one or more regulatory T cell (Treg) stimulators 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 thereof.

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

[0063] In some embodiments, the microparticles include at least one polymer. In further non-limiting examples, the at least one polymer comprises 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-glycolic acid) (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 comprise alginate.

[0065] In other embodiments, the microparticles are about 0.5 μm to 5 μm in diameter and provide sustained release of TGF-β1, rapamycin and / or IL-2. In a specific non-limiting example, the microparticles provide release for about 1 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 includes: 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-chain nuclease, a double-chain 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 a further embodiment, 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 a tolerogenic composition as 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 dissolvable microneedle array; b2) one or more agents that promote tolerogenic DC differentiation; or 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 dissolvable microneedle array comprises: i) a substrate comprising a biocompatible material forming a base; and ii) a plurality of microneedles extending from the base. In further embodiments, the biocompatible material comprises carboxymethyl cellulose, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid (HA), or gelatin. In a specific non-limiting example, the biocompatible material comprises carboxymethyl cellulose.

[0070] In further embodiments, the one or more agents that promote differentiation of tolerogenic dendritic cells include a vitamin or an analog thereof, such as vitamin D3 or a vitamin D3 analog.

[0071] In some embodiments, a method of inducing tolerance to a CRISPR-Cas effector polypeptide in a mammalian subject is disclosed, comprising administering to the subject an effective amount of a tolerogenic composition comprising a microneedle array. In further embodiments, the composition is administered topically, such as intradermally or intradermally.

[0072] In other 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 methods include administering a gene editing composition comprising a CRISPR-Cas effector polypeptide to a subject after administering an effective amount of a tolerogenic composition, 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 administering the tolerogenic composition. The subject may be a human. The subject may be a non-human mammal. CRISPR-Cas effector peptides

[0074] The disclosed methods use 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. A CRISPR-Cas effector polypeptide suitable for use is a Class 2 CRISPR effector polypeptide, also referred to herein as a Class 2 CRISPR-Cas effector polypeptide. 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, for example, a Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide, for example, 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. CRISPR-Cas effector polypeptides suitable for use include CRISPRi polypeptides. See, e.g., Qi et al. (2013) Cell 152:1173; and Jensen et al. (2021) Genome Research doi:10.1101 / gr.275607.121. CRISPR-Cas effector polypeptides suitable for use include CRISPRa polypeptides. See, e.g., Jensen et al. (2021) Genome Research doi:10.1101 / gr.275607.121; and Breinig et al. (2019) Nature Methods 16:51. CRISPR-Cas effector polypeptides suitable for use include CRISPRoff polypeptides. See, e.g., et al. (2021) Cell 184:2503. CRISPR-Cas effector polypeptides suitable for use include nickases. CRISPR-Cas effector polypeptides suitable for use include catalytically inactive CRISPR-Cas effector polypeptides that retain binding to the target nucleic acid (when complexed with the guide RNA). CRISPR-Cas effector polypeptides suitable for use 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 referred to 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 CasY, and homologs thereof. Immunogenic fragments of these CRISPR-Cas effector polypeptides can also be included in the disclosed compositions.

[0075] In some cases, a CRISPR-Cas effector polypeptide suitable for inclusion in a composition of the present disclosure is a Cas9 polypeptide. In some cases, the Cas9 polypeptide comprises Fig.11A The Streptococcus pyogenes Cas9 amino acid sequence shown in (SEQ ID NO: 1) has an amino acid sequence with 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.

[0076] In some cases, the Cas9 polypeptide is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, the saCas9 polypeptide comprises a polypeptide that is identical to any known saCas9 amino acid sequence (e.g., Fig. 11B The saCas9 amino acid sequence shown in (SEQ ID NO: 2)) has an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity.

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

[0078] In some cases, a suitable Cas9 polypeptide comprises a R691A substitution. For example, in some cases, a suitable Cas9 polypeptide comprises Fig.11A The amino acid sequence shown in has an amino acid sequence of at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity, wherein amino acid 691 is Ala.

[0079] In some cases, a suitable Cas9 polypeptide comprises D1135V, R1335Q, and T1337R substitutions. For example, in some cases, a suitable Cas9 polypeptide comprises Figure 1A The amino acid sequence shown in has an amino acid sequence of at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity, wherein amino acid 1135 is Val, amino acid 1335 is Gln, and amino acid 1337 is Arg, and wherein the Cas9 polypeptide exhibits a relaxed PAM requirement.

[0080] In some cases, a suitable Cas9 polypeptide is a SpRY variant. See, e.g., Zhang and Zhang (2020) Trends Genetics 36:546; and Walton et al. (2020) Science 368:290; and U.S. Patent Publication No. 2021 / 0284978. SpRY is a variant of Streptococcus pyogenes Cas9; the variant has a reduced PAM requirement. For example, in some cases, a suitable Cas9 polypeptide comprises D1135L, S1136W, G1218K, E1219Q, R1335Q, and T1337R substitutions. For example, in some cases, a suitable Cas9 polypeptide comprises and 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, a suitable 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 The amino acid sequence shown in has an amino acid sequence of at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity; and comprises amino acid substitutions at one, two, three, four, five or all six of the following positions: at E1219 (e.g., E1219Q, E1219H, E1219S or E1219V substitution); S1136 (e.g., S1136W, S1136F, S1136A or S1136V substitution); D1135 (e.g., D1135L, D1135A, D1135W or D1135F substitution); G1218 (e.g., G1218R, G1218K or G1218S substitution); R1335 (e.g., R1335Q substitution); and T1337 (e.g., T1337R or T1337K substitution).

[0081] In some cases, a suitable Cas9 polypeptide is a Cas9 polypeptide or a Cas9-NG polypeptide. See, e.g., Zhong et al. (2019) Molec. Plant 12:1027; Hu et al. (2018) Nature 556:57; and Nishimasu et al. (2018) Science 361:1259. Cas9 nucleic acid and protein sequences are publicly available. For example, Nucleotides 796693..800799 of accession number CP012045.1 and nucleotides 1100046..1104152 of CP014139.1 disclose Cas9 nucleic acid, and Accession numbers AMA70685.1 and AKP81606.1 disclose Cas9 proteins. In some examples, Cas9 is an inactivated form of Cas9 (dCas9), such as a nuclease-deficient form (e.g., In some examples, the Cas9 has at least 80% sequence identity to such sequences, 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, and retains the ability to cleave DNA.

[0082] In some cases, a suitable CRISPR-Cas effector polypeptide is a V-type CRISPR-Cas effector polypeptide. In some cases, the V-type CRISPR-Cas effector polypeptide is a Cas12a protein. In some cases, the Cas12a protein comprises a polypeptide that is similar to any known Cas12a protein (e.g., Fig. 11C or Fig.11D ), 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.

[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, one or more heterologous polypeptides include single-chain nucleases, double-chain nucleases, helicases, methylases, demethylases, acetylases, deacetylases, deaminases, integrases, recombinases, base editors or guide editors. In some cases, one or more heterologous polypeptides include nuclear localization signals. 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 may also include a suitable carrier.

[0084] CRISPR-Cas effector polypeptides with reduced immunogenicity compared to a parent CRISPR-Cas effector polypeptide are known in the art, 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 in one or more residues corresponding to one or more MHC class I and / or MHC class II binding sites in a wild-type CRISPR-Cas effector polypeptide, wherein 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 may also be used, for example, immunogenic fragments corresponding to MHC class I and / or class II binding sites. The immunogenic fragments may include 9 (9-mers) or 10 (10-mers) amino acids. The immunogenic fragments may 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 may also be used. The mixture may include at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different fragments. These immunogenic fragments may 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 immunogenic fragments thereof, or one or more fusion polypeptides, the mode of administration, the age and weight of the patient. In some cases, higher or lower doses may be appropriate. The administration of the dose may be a single administration in the form of a single dosage unit or several smaller dosage units, or multiple administrations of subdivided doses at specific intervals. A skilled clinician can easily determine the 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, may be included in a pharmaceutically acceptable carrier. These pharmaceutical compositions can be prepared and administered in dosage units. Liquid preparations are generally used. Solid dosage units are tablets, capsules, single injections, and even suppositories. Suitable modes of administration are best determined individually for each subject by a medical practitioner. Various pharmaceutically acceptable carriers and their preparations are described in standard preparation monographs, for example, Remington's Pharmaceutical Sciences by EW Martin. See also Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988. The dosage form of the pharmaceutical composition will be determined by the selected mode of administration. Typically, the pharmaceutical composition includes an effective amount of a CRISPR-Cas effector polypeptide, an immunogenic fragment, and a fusion polypeptide.

[0088] Suitable solid or liquid pharmaceutical preparations are in the form of, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injection solutions in the form of ampoules, and preparations with extended release of one or more CRISPR-Cas effector polypeptides or their immunogenic fragments or one or more fusion polypeptides comprising CRISPR-Cas effector polypeptides, in which excipients and additives and / or adjuvants are generally used, such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, solubilizers or stents. Pharmaceutical compositions are suitable for a variety of drug delivery systems. For a brief review of current drug delivery methods, 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 can be used with other systems (e.g., zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), etc.). particle

[0090] Provided are tolerogenic compositions comprising one or more microparticles, one or more Treg stimulators 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 stimulators include CC chemokine motif (CCL22), interleukin 2 (IL-2), rapamycin, transforming growth factor β (TGF-β), retinoic acid, and vasoactive intestinal peptide (VIP).

[0091] In certain embodiments, Treg stimulators can be very effective in recruiting Tregs. In particular, CCL22, retinoic acid and VIP recruit Tregs. In certain embodiments, microparticles loaded with therapeutic agents can be very effective in recruiting Tregs. Microparticles can effectively recruit endogenous Treg cells to local sites.

[0092] In certain embodiments, Treg stimulators can be very effective in inducing Tregs. In particular, IL-2, rapamycin and TGF-β can effectively induce Tregs. In certain embodiments, microparticles loaded with therapeutic agents can be very effective in stimulating Tregs. Dissolvable microspheres can effectively stimulate endogenous Treg cells at local sites.

[0093] In certain embodiments, without being bound by theory, by starting from CD4 +This approach uses the body’s own natural mechanism to induce tolerance through a subset of antigen presenting cells, called tolerogenic dendritic cells (tDCs), to induce peripheral naive CD4 + T cells differentiate into Tregs. Specifically, tDCs can induce Treg differentiation by secreting IL-2 and TGF-β cytokines, but the maintenance of Tregs is more complicated and depends on the local microenvironment, which is not only conducive to Treg differentiation, but also unfavorable for its differentiation into other effector T cells. One way to ensure that cells do not differentiate into pathogenic effector T cells is through a small molecule, rapamycin. Rapamycin (Rapa) is an mTOR inhibitor that can inhibit the generation and proliferation of effector T cells. Rapamycin derivatives can also be used and are known in the art.

[0094] In certain embodiments, the body's own endogenous Tregs are enriched by delivering a combination of Treg inducers via TRI microspheres (TGF-β1, rapamycin (Rapa) or a derivative thereof, and IL-2), which are microparticles that encapsulate TGF-β1, Rapa, and IL-2. In some embodiments, TGF-β1, Rapa, and IL-2 are included in separate microparticles and are administered as a mixture. Therefore, in some embodiments, the tolerogenic composition comprises i) microparticles comprising TGFβ; ii) microparticles comprising IL-2, and iii) microparticles comprising rapamycin (or a derivative thereof). Without being bound by theory, this system is capable of increasing the prevalence of Tregs, thereby inducing tolerance.

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

[0096] In certain embodiments, the amount of one or more Treg stimulators loaded into one or more microparticles can range from about 1 ng to about 1 mg, more particularly about 1 ng to about 100 μg of reagent per mg of microparticles. In certain specific embodiments, the amount of one or more Treg stimulators loaded into one or more microparticles is about 25 ng-7.5 μg of reagent per mg of microparticles. The Treg stimulator can be IL-2 or TGF-β1. The Treg stimulator can be included in separate microparticles or in one microparticle (which includes a variety of Treg stimulators). As used herein, the administration of "microparticles" refers to the administration of one or more microparticles including a Treg stimulator.

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

[0098] In other embodiments, the microparticles have a diameter of about 0.5 μm to 5 μm and provide sustained release of TGF-β1, rapamycin and / or IL-2. In a specific example, the microparticles provide release for about 1 week.

[0099] The microparticles include at least one polymer. The polymers used for the microparticles can be bioerodible polymers as long as they are biocompatible. Bioerodible polymers include polyhydroxy acids, such as polylactic acid and copolymers thereof. Exemplary polymers include polyglycolide, polylactic acid (PLA) and poly (lactic acid-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 alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylates and methacrylates, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, triacetate Cellulose, sodium 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 may include alginate. In other non-limiting examples, the polymer is ester-terminated PLGA.

[0100] In more embodiments, the polymer is polyethylene glycol-poly(lactic acid-co-glycolic acid). In further embodiments, at least one polymer comprises 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-glycolic acid) (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" the hydrophilicity or hydrophobicity of the polymer to the agent to be encapsulated. In some cases, such as PLGA, this can be achieved by selecting the monomer ratio so that the copolymer has a higher hydrophilicity (for hydrophilic drugs), or has a lower hydrophilicity (for hydrophobic drugs). Alternatively, the polymer can be made to have a higher hydrophilicity, for example, by introducing a carboxyl group on the polymer. In a non-limiting example, a combination of a hydrophilic drug and a hydrophobic drug can be encapsulated in a microparticle prepared from a blend of a 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 about 10kD to about 80kD, more preferably about 10kD to about 35kD. The molecular weight range of PLA is about 20kDa to about 30kDa. The ratio of lactide to glycolide is 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 is 50:50, M n =10 kDa, acid-terminated, referred to as 502H); poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 ratio of lactic acid to glycolic acid, M n =25 kDa, acid-terminated, referred to as 503H); poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 ratio of lactic acid to glycolic acid, M n =30 kDa, acid-terminated, referred to as 504H); poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 ratio of lactic acid to glycolic acid, M n =35 kDa, ester terminated, referred to as 504); and poly(D,L-lactic-co-glycolic acid) (PLGA, lactic acid to glycolic acid ratio of 75:25, M n =10 kDa, referred to as 752).

[0103] The diameter of the microparticles can range from about 0.1 μm to about 1000 μm, or any range therebetween, for example, 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 diameter range of the microparticles is about 0.1 μm to 1 μm, for example, 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 diameter range of the microparticles is about 0.1 μm to 5 μm, for example, 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 volume average diameter of the microparticles loaded can be 200 nm to 30 μm, more particularly 1 μm to 10 μm. In certain embodiments, the volume average diameter of the microparticles loaded with reagents is not 10 μm or larger. The diameter of the microparticles can be, for example, about 15 μm to 30 μm, for example, a diameter of about 15 μm, 20 μm, 25 μm or 30 μm. The diameter of the microparticles can be, for example, about 0.5 μm to 5 μm, such as about 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, or 5 μm in diameter. For example, the diameter of the microparticles can be about 2 μm. The reagent-loaded microparticles can be non-porous, or they can contain varying amounts of pores of varying sizes, typically controlled during synthesis by addition of NaCl.

[0104] The method of manufacturing the agent-loaded microparticles can be either a single emulsion process or a double emulsion process, depending on the desired solubility of the encapsulated agent in water, the molecular weight of the polymer chain used to prepare the microparticles (MW can range from ~1000 Da to over 100,000 Da), which controls the degradation rate of the microparticles and the subsequent drug release kinetics.

[0105] The microparticles used in the methods disclosed herein can provide sustained release. For example, sustained release can last for at least one day, more particularly at least 5 days or at least 10 days, most particularly at least 30 days. The release of the agent can be linear or non-linear (single or multiple burst releases). In certain embodiments, the agent can be released without a burst effect. For example, sustained release can show a substantially linear release rate of the therapeutic agent in vivo for at least one day, more particularly at least 5 days or at least 10 days, most particularly at least 30 days. A substantially linear release rate means that the release rate of the therapeutic agent changes by no more than about 20% over the desired time period, more typically no more than about 10%. It is desirable to provide a relatively constant release rate of the agent from the delivery system during the entire service life of the system. For example, it is desirable that the release amount of the agent is 0.1 μg to 100 μg per day, more particularly 1 μg to 10 μg per day, during the entire service life of the system. However, depending on the formulation composition of the polymer microparticles, the release rate may change (increase or decrease).

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

[0107] The microparticles used in the methods disclosed herein can provide the sustained release of the reagent. As used herein, the term "slow release" or "controlled release" refers to the escape of any attached or encapsulated reagent at a predetermined rate. For example, the sustained release of the reagent can be caused by the predictable biodegradation of the polymer particles (i.e., for example, artificial antigen presenting cells). The biodegradation rate can be predetermined by changing the polymer composition and / or the ratio comprising the particles. Therefore, sustained release can be short-term, or controlled release can be long-term.

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

[0109] In one embodiment, long-term release is between 24 hours and 36 hours. In one embodiment, long-term release is between 3 days and 7 days. In one embodiment, long-term release is between 7 days and 1 month. In some embodiments, microparticles provide a sustained release of one or more Treg stimulants, for example, 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, long-term release is about 1 week, for example, about 2 days to about one week, about 3 days to about one week, about 4 days to about one week, about 5 days to about one week, or about six days to about one week. Long-term release can last less than about one month.

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

[0111] As disclosed herein, tolerogenic compositions (including microparticles) may include excipient components, such as effective amounts of buffers, and antioxidants for protecting them from the effects of ionizing radiation during sterilization. Suitable water-soluble buffers include, but are not limited to, alkali metal and alkaline earth metal carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates, etc., such as sodium phosphates, sodium citrates, sodium borates, sodium acetate, sodium bicarbonate, sodium carbonate, etc. These reagents are advantageously present in an amount sufficient to maintain the system pH between about 2 to about 9, and more preferably about 4 to about 8. Therefore, the buffer may account for about 5 weight percent of the total system. Suitable water-soluble preservatives include sodium bisulfite, sodium bisulfate, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, parabens, methyl parabens, polyvinyl alcohol, benzyl alcohol, phenylethyl alcohol, etc., and mixtures thereof. These agents may be present in an amount of 0.001 wt % to about 5 wt %, and preferably 0.01 wt % to about 2 wt %.

[0112] In certain embodiments, the microparticles disclosed herein may be administered by injection. Injection sites include, but are not limited to, intradermal, subdermal, subcutaneous, or intramuscular administration. In some embodiments, the tolerogenic composition comprising microparticles is delivered by intravenous administration. Microneedle array

[0113] CRISPR-Cas effector polypeptides, immunogenic fragments thereof, or fusion polypeptides can be administered in 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-loaded microarray, which can be prepared using a micro-grinding master mold and a spin-mold, see U.S. Patent Application Publication No. US-2016-0271381-A1. As disclosed in PCT Publication No. WO 2020 / 232394, undercut or anchoring features can improve skin retention during application and can be achieved without interfering with processing steps, thereby allowing MNAs to be removed directly from the mold. Examples of the utility of the microneedle device include, for example, (1) simultaneous delivery of a 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, and optionally other agents to generate a tolerogenic immune response, and (2) topical skin delivery.

[0114] Dissolvable microneedle arrays enable efficient and safe delivery to skin and mucosal surfaces. Fully dissolvable microneedle array substrates and unique microneedle geometries can be utilized to enable efficient delivery of CRISPR-Cas effector polypeptides or immunogenic fragments thereof, or fusion polypeptides comprising CRISPR-Cas effector polypeptides or immunogenic fragments thereof. This technology can also uniquely enable simultaneous co-delivery of multiple chemically distinct agents, thereby enabling multifunctional drug delivery. The agents can promote the differentiation of tolerogenic dendritic cells, for example by inducing hyporesponsiveness to subsequent antigen exposure, or induce T cell differentiation 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:

[0116] Calcipotriene (also known as calcipotriol or MC-903) is a vitamin D3 analog known in the art. Calcipotriene has the following structure: See, e.g., Masuda et al. (1994) J. Biol. Chem. 269:479.

[0117] Alfacalcidol can also be used. Alfacalcidol has the following structure:

[0118] Tacalcitol can also be used. Tacalcitol has the following structure:

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

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

[0121] The microneedle can be preformed to have a shape comprising a first cross-sectional dimension at the top, a second cross-sectional dimension at the bottom, and a third cross-sectional dimension at the middle portion, wherein the middle portion is located between the top and the bottom, and the third cross-sectional dimension is greater than the first cross-sectional dimension and the second cross-sectional dimension.

[0122] In other embodiments, each microneedle comprises multiple layers of soluble biocompatible materials. In some embodiments, various active components are incorporated into the needle tip using manufacturing techniques, see U.S. Patent Application Publication No. US-2016-0271381-A1, which is incorporated herein by reference. Therefore, by concentrating the active components in this way, the remainder of the microneedle array volume includes a low-cost inactive and generally considered safe matrix material. Based on (1) reducing the waste of non-deliverable active components incorporated into the non-needle portion of the microneedle array, and (2) the drug concentration in the needle tip that penetrates the skin is higher, the end result is a greatly improved efficiency of drug delivery.

[0123] Thus, in some embodiments, the active component is concentrated in the microneedle tips of each array. Thus, compared to conventional microneedle arrays, the concentration of the active component is not uniform throughout the microneedle array because there is little or no active component in the supporting base structure. In addition, in some embodiments (e.g., as disclosed in U.S. Patent Application Publication No. US-2016-0271381-A1), the active component is concentrated in the microneedle tips of each array. Figure 3 A. Figure 3 B. Figure 4A and Figure 4BIn some embodiments, the active component is concentrated in the upper half of each microneedle in the array. The active component is concentrated at the tip of the microneedle, which is defined by the area where the microneedle extends from the base in a narrowing and / or tapering manner. The base, in turn, extends from the support structure of the array.

[0124] As described above, in some embodiments, a single microneedle may contain an active component only in the upper half of the microneedle. In other embodiments, a single microneedle may contain an active component only in the tip or in the narrowed portion near the tip of the microneedle. In other embodiments, a single needle may contain an active component 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 also be delivered as disclosed in PCT Application No. PCT / US2016 / 057363 (which is incorporated herein by reference). The PCT application discloses a microneedle array that can be configured to penetrate the stratum corneum to deliver its payload (e.g., a biologic or a bioactive component) 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 therefore be used for skin delivery. Therefore, in some embodiments, the microneedle array includes pyramidal CMC-microneedles. In other embodiments, the microneedle array includes obelisk-shaped needles.

[0126] In order to construct a microneedle array, a substrate can be used to form a portion of each microneedle containing a bioactive component and a portion that does not contain a bioactive component. As described above, each microneedle can contain a bioactive component only in the microneedle, or in some embodiments, only in the upper half of the microneedle, or in other embodiments, only in the portion that tapers gradually near the tip of the microneedle. Therefore, in order to control the delivery of bioactive components and control the cost of the microneedle array, each microneedle can have a portion containing a bioactive component (immunogen and / or adjuvant) and a portion that does not contain a bioactive component. In the embodiments described herein, the portion that does not contain a bioactive component includes the support structure of the microneedle array, and in some embodiments, includes the base (e.g., lower half) of each microneedle in the array.

[0127] In some embodiments, the dissolvable microneedle array includes wherein the dissolvable microneedle array comprises: i) a substrate comprising a biocompatible material, which forms a base; and ii) a plurality of microneedles extending from the base. Various biocompatible materials can be used as the substrate of the microneedle array.

[0128] A 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 a biocompatible material, for example, through a disulfide bond. In some cases, the biocompatible material can be carboxymethyl cellulose. These connections can be cleaved by enzymes in vivo, and / or cleaved 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 a biocompatible material. The bioactive components can include, but are not limited to, agents that promote tolerogenic DC differentiation, CRISPR-Cas effector polypeptides, immunogenic fragments thereof, or fusion polypeptides.

[0129] The structural matrix of biodegradable solid microneedles most commonly includes preparations based on poly(lactic-co-glycolic acid) (PLGA) or carboxymethyl cellulose (CMC); however, other bases may also be used. Other polymer materials include polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid (HA), gelatin and / or mixtures of two or more thereof. Other small molecule excipients may also be included, such as non-reducing sugars (e.g., trehalose, sucrose, etc.). PLGA is a water-insoluble biodegradable polymer, while CMC, PVP, PVA, HA and gelatin are soluble in water.

[0130] In some embodiments, the biocompatible material is CMC. Due to the relatively high temperatures required for manufacturing (e.g., 135 degrees Celsius or higher) and vacuum and / or organic solvents, PLGA-based devices may limit drug delivery and protein delivery applications. In contrast, CMC-based matrices can be formed at room temperature by a simple spin casting and drying process, which makes CMC-microneedle arrays more suitable for adding CRISPR-Cas effector polypeptides or immunogenic fragments thereof, or fusion polypeptides comprising CRISPR-Cas effector polypeptides or immunogenic fragments thereof.

[0131] CMC-hydrogel can be prepared by low viscosity CMC sodium salt in sterile dH2O with or without active ingredients (as described below). In an exemplary embodiment, CMC can be mixed with sterile distilled water (dH2O) and active ingredients to reach a CMC concentration of about 25wt%. The resulting mixture can be stirred to uniformity and balanced at about 4 degrees Celsius for 24 hours. During this period, CMC and any other components can be hydrated and form a hydrogel. The hydrogel can be degassed in a vacuum for about one hour and then centrifuged at about 20,000g for one hour to remove residual microbubbles that may interfere with the CMC-microneedle array spin casting / drying process. The dry matter content of the hydrogel can be tested by drying a portion of the hydrogel (10g) at 85 degrees Celsius for about 72 hours. Ready-to-use CMC-hydrogel is ideally stored at about 4 degrees Celsius until use.

[0132] The active component (e.g., a CRISPR-Cas effector polypeptide, an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof) and optionally another agent (e.g., vitamin D or a vitamin D analog) can be incorporated into the CMC hydrogel at a relatively high (20-30%) CMC-dry biological agent weight ratio prior to the spin casting process. The array can be spin cast at room temperature, making the process compatible with the functional stability of a wide range of bioactive components. Since the master mold and production mold can be reused for a large number of manufacturing cycles, the manufacturing cost can be greatly reduced. The resulting dehydrated CMC-microneedle array is typically stable at room temperature or slightly lower temperatures (e.g., about 4 degrees Celsius) and retains the activity of the added biological agent, facilitating simple, low-cost storage and distribution.

[0133] In an exemplary embodiment, the surface of the production mold can be covered with about 50 μl (for a mold of 11 mm diameter) of CMC-hydrogel and spin-casted by centrifugation at 2,500 g for about 5 minutes. After the initial CMC-hydrogel layer, another 50 μl of CMC-hydrogel can be laid over 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, the excess material at the edges trimmed, collected and stored at about 4 degrees Celsius. The production mold can be cleaned and reused for subsequent casting of microneedle arrays.

[0134] In some embodiments, the CMC-solid can be formed with a layer without active ingredients and a layer with active ingredients. PCT Application No. PCT / US2016 / 057363 Figures 11A-11D (incorporated herein by reference) shows CMC-solids with different shapes (PCT Application No. PCT / US2016 / 057363 Fig.11A and Fig. 11B) and an active load embedded in the upper layer, which becomes part of the microneedle with active components after micro-grinding. PCT / US2016 / 057363 Fig.12 A and Fig.12 B also shows CMC solids with different shapes, among which PCT / US2016 / 057363 Fig.12 B shows a square, and Fig.12 B shows a rectangle. Both CMC solids can be ground to the sizes described herein for further processing. It should be understood that the geometric shapes are not intended to be limiting. Any geometric shape can be used in the methods disclosed herein.

[0135] Typically, the microneedles have mechanical strength to remain intact when inserted into the skin, when retained in place for many days, and when removed for delivery. The microneedles can have a straight cylindrical shaft or a tapered shaft. In one embodiment, the diameter of the microneedle is largest at the base end of the microneedle and tapers at the end away from the base. The microneedles can also be made to have shafts that include both a straight cylindrical (non-tapered) portion and a tapered portion.

[0136] The microneedle can be formed by an axis having a perpendicular cross section to a circular cross section, or the cross section can be non-circular. For example, the cross section of the microneedle can be polygonal (e.g., star-shaped, square, triangle), rectangular or elliptical (oblong), or other shapes. The axis can have one or more holes. The cross-sectional size is generally about 10nm to 1mm, such as 1μm to 200μm, more preferably 10μm to 100μm. The outer diameter is generally about 10μm to about 100μm, and the inner diameter is generally about 3μm to about 80μm.

[0137] The length of the microneedle is generally 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. Depending on the specific application, the length is selected taking into account the inserted portion and the uninserted portion. The microneedle array can include a mixture of microneedles having, for example, various lengths, outer diameters, inner diameters, cross-sectional shapes, and microneedle spacings.

[0138] The microneedles can be oriented perpendicular to the support structure of the microneedle array or at an angle thereto. Preferably, the microneedles are oriented perpendicular to the support structure so that a greater microneedle density per unit area of ​​substrate can be provided. The microneedle array can include a mixture of microneedle orientations, heights or other parameters.

[0139] In some cases, the support structure of the microneedle array is generally about 50 mm in area. 2 Up to 150mm 2 (For example, about 100mm2 Up to 150mm 2 、Approx. 75mm 2 Up to 100mm 2 、About 50mm 2 Up to 75mm 2 、About 50mm 2 Up to 65mm 2 , or about 60mm 2 Up to 65mm 2 )'s base.

[0140] In some cases, the substrate and / or microneedles and other components are formed of flexible materials 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. Flexible devices can promote more consistent penetration during use, as penetration may be limited by deviations in the attachment surface. For example, the surface of human skin is not flat due to dermatoglyphics (i.e., microscopic wrinkles) and hair.

[0141] In some cases, the tolerogenic composition comprising a microneedle array is administered intradermally. In some cases, the tolerogenic composition comprising a microneedle array is administered subdermally. In some cases, the tolerogenic composition comprising a microneedle array is administered subcutaneously. Methods for inducing tolerance

[0142] The present disclosure provides a method of inducing tolerance to CRISPR-Cas effector polypeptides in a mammalian subject, the method comprising administering to the subject an effective amount of a 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, which are 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 involves administering to a subject in need thereof an effective amount of a tolerogenic composition of the present disclosure.

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

[0144] Subjects suitable for treatment with the methods of the present disclosure include subjects in need of gene therapy or subjects to be administered gene editing compositions, for example for the treatment of disease or as antiviral, antipathogen or anticancer therapy, or for biological research. The subject may be a newborn, an adolescent or an adult. Of particular interest are mammalian subjects. Mammalian species that can be treated with the present method include canines and felines; equines; bovines; ovines, etc., as well as 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 studies. In some non-limiting examples, the subject is a human. In other non-limiting examples, the subject is a veterinary subject, including a non-human primate. In some embodiments, the subject may have previously undergone a gene editing procedure, such as using the same or different CRISPR-Cas effector polypeptides or immunogenic fragments thereof, or fusion polypeptides comprising CRISPR-Cas effector polypeptides or immunogenic fragments thereof. These subjects can be selected for treatment.

[0145] In some embodiments, the effective tolerogenic composition, at one or more doses, stimulates Tregs in a subject, such as by inducing Tregs or recruiting Tregs. In some cases, the effective tolerogenic composition at one or more doses increases the number of Tregs in a subject. + 、FOXP3 + and CD25 + Treg and / or CD4 + FoxP3 – IL-10 + Tr1 cells can inhibit reactive T cells. An effective tolerogenic composition in one or more doses can act on naive T cells, including differentiation into Tregs. In some cases, an effective amount of a tolerogenic composition is an amount that increases the number of Tregs 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 times, at least 2.5 times, or more than 2.5 times when administered to a subject in need at one or more doses, compared to the number of Tregs in the subject before treatment with the tolerogenic composition (as determined using an assay described herein or other assays known to those skilled in the art).

[0146] In some cases, an effective amount of the tolerogenic composition in one or more doses increases the number of tolerogenic DCs in a subject. In some cases, an effective amount of a tolerogenic composition is an amount that, when administered to a subject in need thereof in one or more doses, increases the number 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 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 DC. The immune tolerance inducing ability of tolerogenic DC can be assessed using techniques known to those skilled in the art. Various assays known in the art can be used to assess whether the tolerogenic DC described herein induces immune tolerance. In one aspect, the tolerogenic DC described herein creates an anti-inflammatory environment by increasing the secretion of anti-inflammatory cytokines (e.g., IL-10) and reducing the secretion of pro-inflammatory cytokines (e.g., IL-12p70, IL-6, TNFα), thereby inducing immune tolerance. In some cases, ELISA assays or Luminex xMAP assays are used to assess the ability of tolerogenic DC to secrete IL-10, IL-12p70, IL-6, and TNFα. In some cases, the effective amount of the tolerogenic composition in one or more doses increases the immune tolerance-inducing capacity of the tolerogenic DCs in the 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 greater than 2.5-fold compared to the immune tolerance-inducing capacity of the tolerogenic DCs in the subject prior to treatment with the tolerogenic composition.

[0148] In some embodiments, when administered to a subject in need thereof at one or more doses, the tolerogenic composition improves one or more effects associated with the subject's immune response to the CRISPR-Cas effector polypeptide. In some cases, the tolerogenic composition reduces CD4 + The number of reactive T cells (i.e., CD4 T cells that react to CRISPR-Cas effector peptides) + T cells), which in turn leads to CD8 + In some cases, the tolerogenic composition increases CD4 + The number and / or activity of Tregs, which in turn reduces CD4 + Reactive T cells and / or CD8+ The number and / or activity of T-reactive T cells. These cells can be measured in a sample from a subject.

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

[0150] In some embodiments, the tolerogenic composition of the present disclosure is administered to the subject before administering a gene editing composition comprising a CRISPR-Cas effector polypeptide to the subject. The tolerogenic composition can be administered from about one day to about nine months before the gene editing composition, for example, from about one day to about one week, from about one week to about three weeks, from about three weeks to about one month, from about one month to about two months, from about two months to about four months, from about four months to about six months, or from about six months to about nine months. In one embodiment, the gene editing composition is administered to the subject within about six months of administering the tolerogenic composition.

[0151] In some cases, the method includes administering a gene editing composition comprising a CRISPR-Cas effector polypeptide to the subject prior to administering the tolerogenic composition of the present disclosure. The gene editing composition may be administered one day to nine months prior to the tolerogenic composition, for example one day to one week, one week to three weeks, three weeks to one month, one month to two months, two months to four months, four months to six months, or six months to nine months prior to the tolerogenic composition. In some cases, the gene editing composition is administered to the subject within six months of administering the tolerogenic composition. The gene editing composition may include the same CRISPR-Cas effector polypeptide, or a different but related CRISPR-Cas effector polypeptide. In some embodiments, the gene editing composition has been administered to the subject prior to administering the tolerogenic composition, and then after administering the tolerogenic composition, another gene editing composition is subsequently administered. Thus, in this embodiment, the subject undergoes more than one gene editing procedure.

[0152] In some embodiments, the method comprises administering to the subject a gene editing composition that also comprises a CRISPR-Cas effector polypeptide in addition to a 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 administration may be simultaneous.

[0153] In these embodiments, the gene editing composition may include, but is not limited to, guide RNA (gRNA) and CRISPR-Cas effector polypeptides. CRISPR-Cas effector polypeptides suitable 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 modified according to the needs of the gene editing composition. In some cases, CRISPR-Cas effector polypeptides suitable for inclusion in the gene editing composition include catalytically inactive CRISPR-Cas effector polypeptides that retain binding to the target nucleic acid (when compounded with the guide RNA). In some cases, the gene editing composition includes gRNA or nucleic acids encoding guide RNA. In some cases, the gene editing composition includes nucleic acids encoding sequences to be inserted into the target nucleic acid.

[0154] The attending physician or other qualified medical personnel can determine the appropriate dosage of the disclosed tolerogenic composition based on various clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, specific polypeptide or nucleic acid to be administered, the patient's gender, time and route of administration, general health, and other drugs administered simultaneously. The repetition rate of administration can be determined based on the measured residence time and concentration of the administered agent in the body fluid or body tissue. After successful treatment, it is desirable to have the patient receive maintenance therapy, in which 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 dosage levels can vary depending on the type of tolerogenic composition, route of administration, and sensitivity of the subject 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 boost. The prime and boost may be the same dose, or different doses.

[0156] The frequency of administration of the tolerogenic composition of the present disclosure can vary according to any of a variety of factors (e.g., severity of symptoms, etc.). For example, in some cases, the tolerogenic composition of the present disclosure is administered once a month, twice a month, three times a month, once 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, once every other day (qod), once a day (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 can generally be used at the beginning of treatment. The tolerogenic composition can be administered to a subject in a prime-boost administration strategy of the tolerogenic composition, wherein complete tolerance is induced after the second (boost) administration.

[0157] The duration of administration of the tolerogenic composition of the present disclosure, e.g., the time period for administering the tolerogenic composition of the present disclosure, can vary according to any of a variety of factors (e.g., patient response, etc.). For example, the tolerogenic composition of the present disclosure can be administered for a period of about one day to about one week, about two weeks to about four weeks, about one month to about two months, about two months to about four months, about four months to about six months, about six months to about eight months, about eight months to about 1 year, about 1 year to about 2 years, or about 2 years to about 4 years or longer. The tolerogenic composition of the present invention is administered to a subject using any available method and route suitable for drug delivery, including in vivo and in vitro methods and systemic and local administration routes. Suitable administration methods are listed above.

[0158] Reagent test kit

[0159] Provided herein are kits comprising tolerogenic compositions. The tolerogenic compositions may include a1) microparticles; b1) one or more Treg stimulators encapsulated within 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 may include multiple types of microparticles, each of which encapsulates a different Treg stimulator, or one type of microparticle encapsulates more than one Treg stimulator.

[0160] The tolerogenic composition may include a2) a dissolvable microneedle array; b2) one or more agents that promote tolerogenic DC differentiation; 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 may also include other components to facilitate the specific application of the kit design. For example, the kit may also include a buffer and other reagents routinely used to practice a specific method. The kit may include a gene editing composition comprising a CRISPR-Cas effector polypeptide. The kit may include information that the tolerogenic composition induces tolerance to the CRISPR-Cas effector polypeptide present in the gene editing composition. The gene editing system may include a gRNA for a gene of interest.

[0162] The kit may include a container and a label or package insert on or connected to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be made of a variety of materials (e.g., glass or plastic). In several embodiments, the container may have an access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle) so that a specific amount of reagent can be withdrawn.

[0163] In some embodiments, the label or package insert indicates the use of the composition, such as to induce tolerance. Package inserts typically include instructions in the commercial packaging of a product that contain information about the indications, dosage, contraindications, and / or warnings for the use of the product. The instructional material can be written, electronic (e.g., a computer diskette or compact disk), or visual (e.g., 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 treating various human diseases. The Cas9 nuclease used to delete or insert genes is derived from bacteria commonly exposed to humans (such as Streptococcus pyogenes and Staphylococcus aureus (S.aureus)). Therefore, most people already have pre-existing humoral immunity and cellular immunity to these Cas9 proteins, which is a potential obstacle for safe and effective in vivo gene editing (see Charlesworth et al., Nature Medicine, 2019). Specifically, Cas9-specific memory T cells can attack and kill cells in which Cas9 is delivered or induced to express Cas9 in patients. This can prevent gene editing from being successful and may cause tissue or organ toxicity.

[0166] Methods were developed to measure CD4 and CD8 T cell responses to Cas9 in mice, allowing for the evaluation of methods to reduce immunogenicity. Cas9 peptides were identified that were presented by MHC I and MHC II in specific mouse strains. In addition, methods were used to induce immune tolerance (anergy) to Cas9, see Figure 1A-Figure 1C . Two drug delivery systems teach the immune system to tolerate Cas9, potentially preventing harmful immune responses to Cas9 (which would prevent in vivo gene editing). The first approach uses injection of Cas9 protein with regulatory T cell (Treg) inducing microparticles (TRIMP), which are biodegradable polymer microparticles that provide sustained delivery of TGF-B1, rapamycin, and IL-2 to induce the differentiation of natural suppressive Tregs. The second approach uses microneedles to co-deliver Cas9 protein, for example with a tolerogenic vitamin D3 analog (MC903). Delivery to the skin microenvironment is achieved by a soluble microneedle array (MNA). The disclosed method ultimately promotes an increase in the ratio of regulatory T-cells to inflammatory T-cells, thereby preventing or reducing harmful immune responses to cells expressing or internalizing Cas9 to achieve in vivo CRISPR gene therapy. Example 1 Assay for measuring mouse-associated Cas9 responses

[0167] Two robust immunization protocols and ex vivo restimulation protocols were established to measure SpCas9-specific T cell immunity in C57BL / 6 mice. C57BL / 6 immunocompetent mice were immunized with SpCas9 protein and adjuvant to induce SpCas9-specific CD4 T cells or injected with cells expressing SpCas9 to induce SpCas9-specific CD8 T cells. Similar approaches 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). The immunization was performed twice in vivo in C57BL / 6 mice (prime on day 0 and 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 to Cas9 was observed in animals immunized in the flank ( Figure 2 ).

[0169] To prime SpCas9-specific CD4 T cells, TiterMax TM C57BL / 6 mice were immunized twice with SpCas9 protein formulated with Gold adjuvant (primary immunization on day 0 and booster on day 14). Splenocytes of immunized mice were harvested on day 21 and restimulated in vitro with SpCas9 protein. Mice immunized with SpCas9 alone showed antigen-specific recall responses upon restimulation ( Figure 3 ). Example 2 Two different methods for inducing Cas9 tolerance

[0170] Results of tolerogenic microparticles. Cas9 was co-delivered with microparticles (MPs) containing rapamycin, TGF-β, and IL-2 (Tri-MPs) and injected subcutaneously into the back of the neck of mice. The microparticles provided a slow release of the agents, which induced the expansion of specific Tregs and ultimately provided systemic tolerance to antigen-specific challenges. Figure 4B As shown, expansion of regulatory T cells was observed in the spleen after administration of a mixture of Cas9 and microparticles containing rapamycin, TGF-β, and IL-2 ("Tri-MP Cas9"), but this expansion was not observed when the microparticles did not include rapamycin, TGF-β, and IL-2 ("blank MP Cas9") or the administration did not include Cas9 ("Tri-MP").

[0171] To examine the effect on the immune response to Cas9, mice were pretreated with Tri-MP Cas9 and 7 days later treated with Seven days later, the mouse spleen cells were tested by Elispot assay for the presence of IFN-g-producing T cells when stimulated with Cas9. Pretreatment with Tri-MP Cas9 almost completely suppressed the response of CD4 T cells to Cas9 ( Figure 4A ). Unexpectedly, a diminished response was also observed in mice pretreated with Tri-MP without Cas9, likely due to the presence of residual immunosuppressive factors that were present at the time of Cas9 challenge a week later. Therefore, these procedures were repeated, postponing immunization until 14 days after MP administration. A second immunization was performed a week later to boost the response, and spleen cells were tested a week later using a cytokine bead array to quantify IFN-γ produced in the culture ( Figure 4C ). Pretreatment with TRI-MP mixed with Cas9 almost completely eliminated the T cell response. In contrast, pretreatment with TRI-MP alone or with blank-MP mixed with Cas9 had no statistically significant effect. These results show that co-delivery of Cas9 and TRI-MP can expand Tregs and eliminate Cas9 effector T cell responses.

[0172] Results of tolerogenic microneedles: The second method of tolerance induction involved the application of tolerogenic microneedle arrays (MNA) containing Cas9 or the control protein BSA. Mice received three MNA applications. One week later, Cas9 protein (with They were immunized subcutaneously with adjuvant (prepared with Cas9). One week later, spleen cells from the mice were collected and T cell responses to Cas9 were evaluated by intracellular cytokine staining. MNA application, especially with Cas9, reduced the response to subsequent stimulation ( Figure 5 ). Unexpectedly, the BSA control MNA was almost as effective as the Cas9 MNA. Although unexpected, the results suggest that applying the MNA together with Cas9 induces an immunosuppressive environment, thereby reducing the subsequent immune response to Cas9. Example 3

[0173] Figure 1A-Figure 1C The proposed mechanism by which TRI MP and MC903 MNA promote antigen-specific tolerance induction is shown. For specific Cas9 applications, the Cas9 antigen can be injected subcutaneously with the TRI MP instead of being administered to the skin near the TRI MP injection site.

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

[0175] Mice were sensitized (immunized) to Streptococcus pyogenes Cas9 (SpCas9) antigen by application of Cas9 (antigen) + Poly (I:C) (adjuvant) MNA, or by subcutaneous injection of cells expressing Cas9 (specifically splenocytes). Seven days later, mice were challenged (re-exposed to Cas9 antigen) in the right ear by Cas9 MNA, and ear thickness was measured every day for the next four days. Blank (empty) MNA was applied to the contralateral ear to control for any increase in ear thickness that was not related to Cas9 antigen-specific inflammatory response. Ear swelling associated with delayed hypersensitivity (T-cell mediated) to Cas9 is reported as the change in ear thickness (△) (i.e., Cas9 MNA-treated right ear - blank MNA-treated left ear). Greater ear 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+MC903MNA (day 0, day 3, and day 6). Three days later (day 9), mice were sensitized (immunized) to the Cas9 antigen by applying Cas9+Poly(I:C)MNA. Seven days later, mice were challenged (exposed to the Cas9 antigen again) in the right ear by Cas9 MNA, and ear thickness was measured every day for the next four days. Blank (empty) MNA was applied to the contralateral ear to control any increase in ear thickness that was not related to the Cas9 antigen-specific inflammatory response. Ear swelling associated with delayed hypersensitivity to Cas9 (T- cell mediated) was reported as a change in ear thickness (△) (i.e., right ear treated with Cas9 MNA-left ear treated with blank MNA). Greater ear swelling is consistent with a stronger Cas9-specific T-cell mediated immune response, and pretreatment with Cas9+MC903 MNA (preventive tolerance) prevented / reduced subsequent sensitization, thereby reducing delayed hypersensitivity (ear swelling). Unsensitized mice (grey) had no pre-existing immunity to Cas9 prior to ear challenge with Cas9 MNA. Results are shown in Figure 7 middle.

[0177] Four days after ear challenge (see Figure 7 ), the ear skin draining lymph nodes (DLN) were isolated, and T-cell responses were evaluated by flow cytometry. In this model, Cas9+PolyIC MNA sensitization appears to induce a predominantly Th1-mediated proinflammatory effector T-cell response, and pretreatment with Cas9+MC903 MNA before sensitization significantly reduced the Th1 population and increased the Treg / Th1 and Treg / Teff ratios, see Figure 8A-8C The shift in the ratio of regulatory T-cells to effector T-cells is consistent with the induction of a more tolerogenic immune response.

[0178] In contrast to previous models of preventive tolerance induction, tolerance was induced by treating previously sensitized mice (i.e., mice with pre-existing immunity to Cas9) with Cas9+MC903 MNA, see Figure 9A-9B In this therapeutic desensitization model, mice were first sensitized (immunized) to Streptococcus pyogenes Cas9 (SpCas9) antigens by applying Cas9+Poly(I:C)MNA or subcutaneously injecting splenocytes expressing Cas9. Starting one week later, some mice were treated with Cas9+MC903 MNA (days 7, 10, and 13). Five days after the last treatment with Cas9+MC903 MNA (day 18), all mice received CFSE expressing Cas9. 高 "Target" cells (splenocytes) and "control" CFSE低 Adoptive transfer of a mixture of cells (i.e., spleen cells that do not express Cas9). Target cells were labeled with CFSE dye (10uM), and control cells were labeled with CFSE (1uM), so they could be identified and distinguished by flow cytometry. The next day, mouse spleens were processed and analyzed by flow cytometry to measure specific cell lysis (i.e., specific killing of target cells expressing Cas9 by Cas9-specific effector T cells (especially cytotoxic T cells)). The specific lysis percentage was calculated as

[0179] As expected, the initial Mice did not show specific lysis of target cells expressing Cas9, which is consistent with the lack of Cas9-specific cytotoxic T cells. Mice primed with Cas9+PolyIC MNA also did not show specific lysis, indicating that this sensitization method did not induce a strong (robust) cytotoxic T-cell response. This is consistent with the lack of Cas9-specific cytotoxic T cells. Figure 8A-8C This is consistent with the results showing a predominantly Th1 response in the 247 samples and is likely due to the lack of cross-presentation of exogenous (extracellular protein) antigens to CD8+ T cells. In contrast, subcutaneous injection of splenocytes expressing Cas9 resulted in specific lysis of target cells expressing Cas9 (see Figure 9A-9B ), and treatment with Cas9+MC903 MNA after sensitization slightly reduced the degree of specific cleavage (see Figure 9A-9B ). This in vivo specific cleavage assay is a relevant indicator of tolerance induction and suppression of Cas9-specific T-cell responses, as preventing the immune system from lysing or killing Cas9-expressing cells is the ultimate goal of achieving in vivo editing using CRISPR-Cas9.

[0180] Pretreatment with SpCas9 and Mc903 MNA reduces sensitization and subsequent delayed-type hypersensitivity reactions. C57BL / 6 mice were treated with MC903MNA, SpCas9 MNA, or SpCas9+MC903 MNA (days 0, 3, and 6) and then sensitized with SpCas9+PolyIC MNA (day 9). Control mice were sensitized but tolerance was not induced (pretreatment). Five days after sensitization, DTH reactions were elicited by applying SpCas9 MNA in the right ear. Blank MNA was applied to the contralateral ear, and ear swelling one to four days after challenge was reported as the difference between the ear thickness of the SpCas9 MNA-treated ear and the blank MNA-treated ear. Pretreatment with SpCas9+MC903 MNA reduced ear swelling DTH reactions to SpCas9 challenge, see Fig.12In 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 (demonstrating reduced antigen-specific T-cell-mediated inflammation).

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

[0182] Obviously, the precise details of the methods or compositions may be changed or modified without departing from the spirit of the described invention. We claim all such modifications and variations that come within the scope and spirit of the following claims.

Claims

1. A tolerogenic composition comprising: a1) one or more microparticles; b1) one or more regulatory T cell (Treg) stimulators 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) dissolvable microneedle array; b2) one or more agents that promote differentiation of tolerogenic DCs in the dissolvable 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.

2. The tolerogenic composition according to claim 1, comprising: a1) one or more microparticles; b1) the one or more regulatory T cell (Treg) stimulators 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. 3 . The tolerogenic composition of claim 2 , wherein the one or more Treg stimulators comprise CCL22, retinoic acid, or vasoactive intestinal peptide.

4. The tolerogenic composition of claim 2 or claim 3, wherein the one or more Treg stimulators comprise IL-2, TGF-β, rapamycin, a rapamycin derivative, or a CCR8 ligand.

5. The tolerogenic composition of any one of claims 2-4, wherein the one or more Treg stimulators comprise TGFβ, IL2 and rapamycin.

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

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

8. The tolerogenic composition of claim 7, wherein the at least one polymer comprises polyethylene glycol (PEG), poly(amino acids), 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-glycolic acid) (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.

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

10. The tolerogenic composition of any one of claims 1-9, wherein the one or more microparticles are formulated for sustained release of the one or more regulatory T cell (Treg) stimulators.

11. The tolerogenic composition of any one of claims 1-10, wherein 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.

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

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

14. A method of 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 any one of claims 2 to 13, thereby inducing tolerance to the CRISPR-Cas effector polypeptide.

15. The method of claim 14, wherein the composition is administered intradermally, subdermally, subcutaneously or intramuscularly.

16. The tolerogenic composition of claim 1, wherein the composition comprises: a2) the dissolvable microneedle array; b2) one or more agents that promote differentiation of tolerogenic DCs in the dissolvable microneedle array; c2) CRISPR-Cas effector polypeptide or an immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof.

17. The tolerogenic composition of claim 16, wherein the dissolvable microneedle array comprises: i) a substrate comprising a biocompatible material forming a base; and ii) a plurality of microneedles extending from the base.

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

19. The tolerogenic composition of claim 18, wherein the biocompatible material comprises carboxymethylcellulose.

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

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

22. The method of claim 21, wherein the composition is administered intradermally or subdermally.

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

24. The method of 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: After administering the effective amount of the tolerogenic composition, administering 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.

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

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

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

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

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

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