Il-10 mutants for Anti-inflammatory therapy

Abstract

This disclosure relates to modified IL-10 proteins and rAAVs for delivering a transgene encoding the modified IL-10 proteins to target cells as well as methods of their use.

Description

IL-10 MUTANTS FOR ANTI-INFLAMMATORY THERAPYRELATED APPLICATIONS

[0001] The application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application number 63 / 693,665, filed September 11, 2024, the contents of which are incorporated by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. W81XWH2210065, awarded by the U.S. Army Medical Research Acquisition Activity. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (U119770253WO00-SEQ-GJM,.xml; Size: 22,454 bytes; and Date of Creation: September 11, 2025) are herein incorporated by reference in its entirety.BACKGROUND OF INVENTION

[0004] Interleukin- 10 (IL- 10) is a cytokine that is recognized for its potent anti-inflammatory and immunoregulatory properties. IL- 10 is secreted by multiple immune cell types, including regulatory T cells, macrophages, and dendritic cells, and plays a critical role in limiting excessive immune responses and maintaining immune homeostasis. Because of these properties, IL- 10 has been investigated as a therapeutic candidate for a variety of inflammatory and autoimmune diseases, including inflammatory bowel disease, rheumatoid arthritis, and psoriasis. Despite its therapeutic potential, clinical application of IL- 10 has been limited by its pleiotropic nature. In particular, IL- 10 can exert pro-inflammatory effects by stimulating T effector cells, resulting in increased production of intcrl'cron-v(IFN-v) and cytotoxic effector molecules such as granzyme B, thereby reducing the overall therapeutic benefit of IL- 10 administration. There remains a need in the art for strategies that preserve the anti-inflammatory and immunoregulatory benefits of IL- 10 while minimizing or eliminating its undesirable pro- inflammatory effects.1U1197.70253WO00#1479354.2SUMMARY

[0005] Aspects of the disclosure relate to “modified” or “variant” IL- 10 proteins which preserve the anti-inflammatory and immunoregulatory benefits of wildtype IL- 10 while minimizing or eliminating the undesirable pro-inflammatory effects of wildtype IL- 10. Accordingly, in certain aspects, the disclosure provides modified IL- 10 cytokines which comprise one or more amino acid substitutions relative to a wildtype IL- 10, wherein the modified IL- 10 cytokines preserve the anti-inflammatory and immunoregulatory benefits of a wildtype IL- 10 while minimizing or eliminating the undesirable pro-inflammatory effects of a wildtype IL- 10. In certain embodiments, the variant IL- 10 cytokines can be derived from (e.g. computationally derived from, as exemplified in the Examples) a starting point wildtype IL- 10 cytokine, such as SEQ ID NO: 1. As used herein, the term “derived from” refers to a protein or polypeptide that is based on, originates from, or is modified relative to a reference or wildtype protein sequence. A protein “derived from” a wildtype protein may share substantial sequence identity with the wildtype sequence but can include one or more modifications such as substitutions, deletions, insertions, truncations, or additions. And, due to the one or more modifications, such modified proteins may have altered properties or functions relative to the starting protein, e.g. variants of IL- 10 may retain normal anti-inflammatory effect of a wildtype IL- 10, but have no or lower pro-inflammatory function.

[0006] In various embodiments, the variant IL- 10 cytokines comprise an amino acid sequence that has at least one amino acid substitution, deletion, or insertion relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In various other embodiments, the variant IL- 10 cytokines comprise an amino acid sequence that has at least two amino acid substitutions, deletions, or insertions relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In still other embodiments, the variant IL- 10 cytokines comprise an amino acid sequence that has at least three amino acid substitutions, deletions, or insertions relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In yet other embodiments, the variant IL- 10 cytokines comprise an amino acid sequence that has at least four amino acid substitutions, deletions, or insertions relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In other embodiments, the variant IL-10 cytokines comprise an amino acid sequence that has at least five amino acid substitutions, deletions, or insertions relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In other embodiments, the variant IL- 10 cytokines comprise an amino acid sequence that has at least six amino acid2U1197.70253WO00#1479354.2substitutions, deletions, or insertions relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In other embodiments, the variant IL-10 cytokines comprise an amino acid sequence that has at least seven amino acid substitutions, deletions, or insertions relative to a wildtype IL-10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In other embodiments, the variant IL- 10 cytokines comprise an amino acid sequence that has at least eight amino acid substitutions, deletions, or insertions relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In other embodiments, the variant IL-10 cytokines comprise an amino acid sequence that has at least nine amino acid substitutions, deletions, or insertions relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In other embodiments, the variant IL- 10 cytokines comprise an amino acid sequence that has at least ten or more amino acid substitutions, deletions, or insertions relative to a wildtype IL- 10 cytokine amino acid sequence (e.g. SEQ ID NO: 1). In various embodiments, the amino acid substitutions, insertions, or deletions can be contiguous or non-contiguous.

[0007] In a further aspect, the present disclosure provides nucleic acid molecules encoding variant IL- 10 cytokines which comprise one or more amino acid substitutions relative to a wildtype IL- 10, wherein the modified IL- 10 cytokine preserves the anti-inflammatory and immunoregulatory benefits of wildtype IL- 10 while minimizing or eliminating the undesirable pro-inflammatory effects of wildtype IL- 10. In various embodiments, the one or more nucleic acid molecules can include DNA molecules, RNA molecules, plasmids, cloning vectors, and recombinant viral genomes (e.g. recombinant AAV or LV genomes configured to comprise a nucleic acid sequence that encodes a variant IL- 10 cytokine). In still further aspects, the disclosure relates to viral vectors (e.g., adeno-associated virus vectors or lentivirus vectors) or other delivery systems (e.g. non-viral delivery systems such as lipid nanoparticles (LNP)) which comprise one or more variant IL- 10 cytokines, or one or more nucleic acid molecules encoding said one or more variant IL- 10 cytokines, wherein the modified IL- 10 cytokine preserves the anti-inflammatory and immunoregulatory benefits of wildtype IL- 10 while minimizing or eliminating the undesirable pro-inflammatory effects of wildtype IL- 10. In still other aspects, the compositions of the disclosure (e.g., variant IL-10 cytokines, nucleic acid molecules encoding variant IL- 10 cytokines, and vectors comprising variant IL- 10 cytokines and / or nucleic acid molecules encoding variant IL- 10 cytokines) and a recombinant adeno associated virus (rAAV) encoding the modified IL- 10 proteins. The modified IL- 10 proteins described herein retain normal anti-inflammatory effect, but have no or lower pro -inflammatory effects as compared to wild-type IL- 10 protein.3U1197.70253WO00#1479354.2

[0008] In some aspects, the present disclosure provides a modified IL- 10 protein comprising an amino acid sequence of any one or the sequences described in the Sequence Listing which is part of the present disclosure. In another aspects, the present disclosure provides a modified IL- 10 protein comprising an amino acid sequence of any one or the sequences described in the Sequence Listing which is part of the present disclosure.

[0009] In other aspects, the present disclosure provides a modified IL- 10 protein comprising an amino acid sequence of any one or the sequences described in the section entitled SEQUENCES which is part of the present disclosure.

[0010] In some embodiments, a modified IL- 10 protein can have an amino acid substitution at a position corresponding to D25, R32 and / or E96 of the wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the modified IL- 10 protein comprises a R32D substitution of the wildtype IL- 10 protein of SEQ ID NO: 1. In some embodiments, the modified IL- 10 protein comprises a E96R substitution of the wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the modified IL- 10 protein comprises a D25R substitution of the wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the modified IL- 10 protein comprises a E96R substitution of the wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the modified IL- 10 protein comprises a E96R substitution of the wild-type IL- 10 protein of SEQ ID NO: 1. in some embodiments, the modified IL- 10 protein comprises an amino acid substitution at amino acid R32, wherein the amino acid substitution is a D or E. In some embodiments, the modified IL- 10 protein comprises an amino acid substitution at amino acid E96, wherein the amino acid substitution is a R or K. In some embodiments, the modified IL- 10 protein comprises an amino acid substitution at amino acid D25, wherein the amino acid substitution is A, R, or K. In some embodiments, the modified IL- 10 protein comprises an amino acid substitution at amino acid E96, wherein the amino acid substitution is a R or K. In some embodiments, the modified IL- 10 protein comprises an amino acid substitution at amino acid E96, wherein the amino acid substitution is A, R, or K.

[0011] In some aspects, the present disclosure provides a modified IL- 10 protein comprising an amino acid substitution at a position corresponding to R32D and / or E96R of the wild-type IL- 10 protein of SEQ ID NO: 1.

[0012] In some aspects, the present disclosure provides a modified IL- 10 protein comprising an amino acid sequence selected from SEQ ID NOs: 2-15, wherein:4U1197.70253WO00#1479354.2SEQ ID NO: 2 (also referred to herein as hIL-lOA) comprises a R32D substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2, and which retains the recited substitution(s) of SEQ ID NO: 2;SEQ ID NO: 3 (also referred to herein as hIL-lOB) comprises a E96R substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 3, and which retains the recited substitution(s) of SEQ ID NO: 3;SEQ ID NO: 4 (also referred to herein as hIL-lOC) comprises an R32D and an E96R substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 4, and which retains the recited substitution(s) of SEQ ID NO: 4;SEQ ID NO: 5 comprises a R32E substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 5, and which retains the recited substitution(s) of SEQ ID NO: 5;SEQ ID NO: 6 comprises a E96K substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 6, and which retains the recited substitution(s) of SEQ ID NO: 6;SEQ ID NO: 7 comprises a D25R substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 7, and which retains the recited substitution(s) of SEQ ID NO: 7;SEQ ID NO: 8 comprises a D25K substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 8, and which retains the recited substitution(s) of SEQ ID NO: 8;SEQ ID NO: 9 comprises a R32D and E96K substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least5U1197.70253WO00#1479354.297%, at least 98%, or at least 99% identity with SEQ ID NO: 9, and which retains the recited substitution(s) of SEQ ID NO: 9;SEQ ID NO: 10 comprises a R32E and E96R substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 10, and which retains the recited substitution(s) of SEQ ID NO: 10;SEQ ID NO: 11 comprises a R32E and E96K substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11, and which retains the recited substitution(s) of SEQ ID NO: 11;SEQ ID NO: 12 comprises a D25K and E96R substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 12, and which retains the recited substitution(s) of SEQ ID NO: 12;SEQ ID NO: 13 comprises a D25K and E96K substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 13, and which retains the recited substitution(s) of SEQ ID NO: 13;SEQ ID NO: 14 comprises a D25R and E96R substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 14, and which retains the recited substitution(s) of SEQ ID NO: 14; andSEQ ID NO: 15 comprises a D25R and E96E substitution relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 15, and which retains the recited substitution(s) of SEQ ID NO: 15.

[0013] In some aspects, the present disclosure provides an isolated nucleic acid encoding a modified IL- 10 protein as disclosed herein.

[0014] In some aspects, the present disclosure provides a vector comprising an isolated nucleic acid encoding a modified IL- 10 protein as disclosed herein.6U1197.70253WO00#1479354.2

[0015] In some aspects, the present disclosure provides, a rAAV comprising a vector, comprising an isolated nucleic acid encoding a modified IL- 10 protein as disclosed herein.

[0016] In some aspects, the present disclosure provides, a rAAV comprising a nucleic acid sequence encoding a modified IL- 10 protein, wherein the modified IL- 10 protein comprises an amino acid substitution at a position corresponding to D25, R32 and / or E96 of the wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the rAAV encoding a modified IL- 10 protein, comprises a modified IL- 10 protein comprising a R32D substitution, a E96R substitution, and / or a D25R substitution of the wild-type IL- 10 protein of SEQ ID NO: 1.

[0017] In some embodiments, a pharmaceutical composition comprises the modified IL- 10 protein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises the rAAV and a pharmaceutically acceptable carrier.

[0018] In some aspects, the present disclosure provides, a method of producing a modified IL- 10 protein by contacting a host cell with a rAAV. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell.

[0019] In some aspects, the present disclosure provides, a method of treating inflammatory disorders or autoimmune disorders by administering a subject an effective amount of a modified IL- 10 protein, a rAAV, or a pharmaceutical composition. In some embodiments, disorder is colitis, sepsis, inflammatory bowel syndrome (IBD), rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), Grave’s disease, Sjorgren’s syndrome, Multiple Sclerosis, allergic asthma, asthma, or autoimmune lymphoproliferative syndrome. In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0020] In some aspects, the present disclosure provides, a method of treating a patient by administering a modified IL- 10 protein, the improvement comprising said modified IL- 10 protein, wherein said modified IL- 10 protein retains normal anti-inflammatory effect, but has no or lower pro -inflammatory effects as compared to wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the modified IL- 10 protein is disclosed herein.

[0021] In some aspects, the present disclosure provides, a method of treating a subject by administering a modified IL- 10 protein comprising: (i) means for binding an IL- 10 receptor; and (ii) a modified IL-10 protein as disclosed here, wherein said modified IL-10 protein retains normal anti-inflammatory effect, but has no or lower pro-inflammatory effects as compared to wild-type IL- 10 protein of SEQ ID NO: 1.7U1197.70253WO00#1479354.2

[0022] In some aspects, the present disclosure provides, a method of treating a patient by administering a rAAV vector comprising a nucleic acid sequence encoding a modified IL- 10 protein, the improvement comprising expressing said modified IL- 10 protein, wherein said modified IL- 10 protein retains normal anti-inflammatory effect, but has no or lower pro- inflammatory effects as compared to wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the rAAV is disclosed herein.

[0023] In some aspects, the present disclosure provides, a method of treating a subject by administering a rAAV vector comprising: a transgene encoding a modified IL- 10 protein as disclosed herein, wherein said modified IL- 10 protein retains normal anti-inflammatory effect, but has no or lower pro -inflammatory effects as compared to wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the rAAV is disclosed herein.BRIEF DESCRIPTION OF DRAWINGS

[0024] FIG. 1 shows recombinant adeno associated virus (rAAV) vector constructs encoding wildtype IL- 10 (rAAV-CB-hILlOW), mutant IL- 10 A (rAAV-CB-hILlOA), mutant IL- 10 B (rAAV-CB-hILlOB), and mutant IL- 10 C (rAAV-CB-hILlOC). Abbreviations: AAV inverted terminal repeat sequences, ITR; poly A sequences, PolyA; CMV enhancer chicken-beta actin promoter, CBp.

[0025] FIG. 2 shows western blot analysis measuring pSTAT3 in PBMCs (top) and U-937 cells (bottom) treated with wild type human IL- 10 (WT) and mutant human IL- 10 (A, B, and C).

[0026] FIGs. 3A-3D shows the evaluations of mutant human IL- 10 (hIL-10) and mutant mouse IL- 10 (mIL-10) in human and mouse cell lines. FIG. 3A shows the evaluation of mutant hIL-10 (A, B, and C) versus wildtype hIL-10 (W) in THP-Luc cells. FIG. 3B shows the evaluations of mutant hIL-10 (A, B, and C) versus wildtype hIL-10 (W) in U937-Luc cells. FIG. 3C shows the evaluations of mutant hIL-10 (A, B, and C) versus wildtype hIL-10 (W) in RAW-Luc cells.FIG. 3D shows the evaluations of mutant mIL-10 (X, Y, and Z) versus wildtype mIL-10 (W) in RAW-Luc cells.

[0027] FIG. 4 shows representative levels of human IL- 10 in rAAV vector injected mice in a DSS model of inflammatory bowel syndrome (IBD). Arrow and line indicate time of DSS treatment.8U1197.70253WO00#1479354.2

[0028] FIG. 5 shows representative disease activity index (DAI) scores seven days after the induction of DSS-induced inflammatory bowel syndrome (IBD) in mice which were injected with human wildtype (W) or mutant (A, B, or C) IL- 10 vectors.

[0029] FIG. 6 shows the average spleen weights 14 days after DSS-induced inflammatory bowel syndrome (IBD) in mice which were injected with human wildtype (W) or mutant (A, B, or C) IL- 10 vectors.

[0030] FIGs. 7A-7C shows representative flowcytometry data detecting STAT3 levels (MFI) on T-cells (FIG. 7A), Natural Kill (NK) cells (FIG. 7B), and monocytes (FIG. 7C) isolated from mice injected with human wildtype (W) or mutant (A, B, or C) IL- 10 vectors 14 days after DSS-induced inflammatory bowel syndrome (IBD).

[0031] FIG. 8 shows the relative levels of IL- 10 in IL- 10 knockout (IL- 10 KO) mice transfected with wildtype (W) or mutant (A, B, or C) IL-10.

[0032] FIGs. 9A-9E shows the body weight changes in IL- 10 knockout (IL- 10 KO) mice transfected with wildtype (W) IL- 10 (FIG. 9A and 9E), mutant IL- 10 A (FIG. 9B and 9E), mutant IL- 10 B (FIG. 9C and 9E), mutant IL- 10 C (FIG. 9D and 9E), or GFP (FIGs. 9A-9E).

[0033] FIGs. 10A-10E show disease activity index (DAI) scores in IL- 10 knockout (IL- 10 KO) mice transfected with wildtype (W) IL- 10 (FIG. 10A and 10E), mutant IL- 10 A (FIG. 10B and 10E), mutant IL- 10 B (FIG. 10C and 10E), mutant IL- 10 C (FIG. 10D and 10E), or GFP (FIGs. 10A-10E).

[0034] FIG. 11 show IL- 10 mutants design for IBD. See Example 7.

[0035] FIGs. 12A-12C show in-vitro study of IL- 10 mutants. See Example 7.

[0036] FIGs. 13A-13E show the DSS-induced Mouse Model. See Example 7.

[0037] FIGs. 14A-14E depict the IL-10KO Mouse Study. See Example 7.

[0038] FIGs. 15A-15E show the effects of IL-10 (W, A, B, and C constructs) on pSTAT3 levels in immune cells in the IL-10KO mice. See Example 7.DETAILED DESCRIPTION

[0039] Although IL- 10 is considered as an anti-inflammatory and immunoregulatory cytokine, its therapeutic effect is limited by its pleiotropic nature, the pro -inflammatory effect by stimulating T effector cells to produce interferon- v(IFN-v) and granzyme B (Saraiva et al. 2020;9U1197.70253WO00#1479354.2Chan et al. 2015). These opposing biological effects (anti-inflammatory and pro-inflammatory) are due to the fact that IL- 10 receptor subunits (IL-lORa and IL-10RP) are expressed in both macrophages and CD8+ T cells. While IL-lO’s interaction with its receptors on macrophages can mediate anti-inflammatory effect, the interaction with its receptors on CD8+ T cells and NK cells will mediate pro-inflammatory effects. Interestingly, two factors play important roles in altering these opposing biological effects of IL- 10: 1) the density of the receptor subunits on the target cells; 2) the affinity of IL- 10 to the receptor subunits. It has been shown that the IL-10RP expression in CD8+ cells and NK cells are much lower than that in macrophages (Saxton et al. 2021). In addition, IL- 10 has lower affinity to IL-10RP than to IL-lORa (Ouyang et al. 2011). These differences create a window that IL- 10 may have more anti-inflammatory effect and relatively lower pro-inflammatory effect. Further, recombinant human IL- 10 has been tested in multiple clinical trials for autoimmune diseases and inflammatory disorders, however no clinical therapy using IL- 10 has been approved. It is believed that this is due to the pleiotropic nature of IL- 10.

[0040] As a solution to this problem, described herein are modified IL- 10 proteins (e.g., mutant IL- 10 proteins) which retain normal anti-inflammatory effect, but have no or lower pro- inflammatory effects. In various aspects, the modified IL- 10 proteins are useful in the treatment of inflammatory and / or autoimmune disorders, such as, but not limited to colitis, sepsis, inflammatory bowel syndrome (IBD), rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), Grave’s disease, Sjorgren’s syndrome, Multiple Sclerosis, allergic asthma, asthma, or autoimmune lymphoproliferative syndrome. The proteins disclosed herein may be used in a variety of applications including, but not limited to compositions and methods (e.g., therapeutic methods). Therapeutic methods disclosed herein include those useful in the treatment of diseases (e.g., inflammatory disorders and / or autoimmune disorders), in subjects in need thereof. Delivery of the modified IL- 10 encoding gene to cells was achieved by use of AAV particles as carriers.

[0041] Accordingly, aspects of the present disclosure relate to “modified” or “variant” IL- 10 proteins that retain the anti-inflammatory and immunoregulatory properties of wild-type IL- 10 while reducing or eliminating undesirable pro -inflammatory effects. In certain embodiments, the modified IL- 10 cytokines comprise one or more amino acid substitutions relative to a wild-type IL-10 (e.g., SEQ ID NO: 1) and can be computationally designed, as exemplified herein.10U1197.70253WO00#1479354.2

[0042] In further aspects, the disclosure provides nucleic acid molecules encoding the modified IL- 10 cytokines described herein. Such nucleic acids may include DNA, RNA, plasmids, cloning vectors, and recombinant viral genomes (e.g., rAAV or lentiviral genomes) encoding a variant IL-10 cytokine. Additional embodiments include viral vectors (e.g., adeno-associated virus (AAV) or lentivirus vectors) and non-viral delivery systems (e.g., lipid nanoparticles) comprising either (i) one or more variant IL- 10 cytokines or (ii) one or more nucleic acid molecules encoding the same. In embodiments, the modified IL- 10 cytokines maintain the beneficial anti-inflammatory activity of wild-type IL- 10 but exhibit reduced or absent pro- inflammatory activity.

[0043] In some embodiments, the modified IL- 10 protein comprises an amino acid sequence selected from any one of the sequences provided in the Sequence Listing, which forms part of this disclosure, including SEQ ID NOs: 2-29.

[0044] In additional embodiments, the modified IL- 10 protein comprises an amino acid sequence selected from any one of the sequences described in the section entitled “SEQUENCES,” including SEQ ID NOs: 2-29.

[0045] In certain embodiments, the modified IL- 10 protein comprises one or more amino acid substitutions at positions corresponding to D25, R32, and / or E96 of SEQ ID NO: 1. Nonlimiting examples include:(a) R32D or R32E substitutions;(b) E96R, E96K, or E96A substitutions;(c) D25A, D25R, or D25K substitutions;(d) combinations of said substitutions, including but not limited to a modified IL- 10 protein have D25A / D25R / D25K and R32D / R32E substitutions, D25A / D25R / D25K and E96R / E96K / E96A substitutions, and R32D / R32E and E96R / E96K / E96A substitutions;(e) combinations of said substitutions, including but not limited to a modified IL- 10 protein have D25X and R32X paired substitutions, D25X and E96X paired substitutions, and R32X and E96X paired substitutions, wherein “X” is any amino acid substitution other than the wildtype amino acid at the corresponding position in SEQ ID NO: 1;(f) combinations of said substitutions, including but not limited to a modified IL- 10 protein have D25X and R32X and E96X triplet substitutions, wherein “X” is any amino acid11U1197.70253WO00#1479354.2substitution other than the wildtype amino acid at the corresponding position in SEQ ID NO: 1; and wherein said modified IL- 10 proteins maintain normal anti-inflammatory activity while exhibiting no or reduced pro-inflammatory activity compared to wild-type IL- 10.Definitions

[0046] As used herein, “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed.

[0047] As used herein, “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region and a light chain constant region. The VH and VL regions retain the binding specificity to the antigen and can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR). The CDRs are interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four framework regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0048] As used herein, “anti-infective” refers to compounds or molecules that can either kill an infectious agent or inhibit it from spreading. Anti-infectives include, but are not limited to, antibiotics, antibacterials, antifungals, antivirals, and antiprotozoans.

[0049] As used herein, "composition" refers to a combination of active agent and at least one other compound or molecule, inert (for example, a detectable agent or label) or active, such as an adjuvant.

[0050] As used herein, “concentrated” refers to a molecule, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than that of its naturally occurring counterpart.12U1197.70253WO00#1479354.2

[0051] As used herein, “control” is an alternative subject or sample used in an experiment for comparison purpose and included to minimize or distinguish the effect of variables other than an independent variable.

[0052] As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” generally refer to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.

[0053] As used herein, “DNA molecule” includes nucleic acids / polynucleotides that are made of DNA.

[0054] As used herein, “diluted” refers to a molecule, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is less than that of its naturally occurring counterpart.

[0055] As used herein, “dose,” “unit dose,” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the targeted effector fusion protein, a composition containing the targeted effector fusion protein, and / or a pharmaceutical formulation thereof calculated to produce the desired response or responses in association with its administration.

[0056] As used herein, “effective amount” is an amount sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human. An effective amount can be administered in one or more administrations, applications, or dosages. The term also includes within its scope amounts effective to enhance normal physiological function. “Effective amount” can refer to the amount sufficient to modulate the immune system in a subject and / or treat and / or prevent an inflammatory disease and / or a symptom thereof in a subject.

[0057] As used herein, “identity,” is a relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome 13U1197.70253WO00#1479354.2Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994;Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested.Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 1970, 48: 443-453,) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure.

[0058] As used herein, “immunomodulator,” refers to an agent, such as a therapeutic agent, which is capable of modulating or regulating one or more immune function or response.

[0059] As used herein, “mammal,” for the purposes of treatments, refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as, but not limited to, dogs, horses, cats, and cows.

[0060] As used herein, “matrix” refers to a material, in which one or more specialized structures, molecules, or compositions, are embedded.

[0061] The term “molecular weight”, as used herein, generally refers to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number-average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

[0062] As used herein, “negative control” refers to a “control” that is designed to produce no effect or result, provided that all reagents are functioning properly and that the experiment is properly conducted. Other terms that are interchangeable with “negative control” include “sham,” “placebo,” and “mock.”14U1197.70253WO00#1479354.2

[0063] As used herein, “nucleic acid” and “polynucleotide” generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double- stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple -helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or "polynucleotide" as that term is intended herein.

[0064] As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.

[0065] As used herein, "organism", "host", and "subject" refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single isolated eukaryotic cell or cultured cell or cell line, or as complex as a mammal, including a human being, and animals (e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans). "Subject" may also be a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0066] As used herein, “patient” refers to an organism, host, or subject in need of treatment.15U1197.70253WO00#1479354.2

[0067] As used herein “peptide” refers to chains of at least 2 amino acids that are short, relative to a protein or polypeptide.

[0068] As used herein, “pharmaceutical formulation” refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.

[0069] As used herein, “pharmaceutically acceptable carrier or excipient” refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, nontoxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.

[0070] As used herein, “pharmaceutically acceptable salt” refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.

[0071] As used herein, “plasmid” as used herein refers to a non-chromosomal double- stranded DNA sequence including an intact “replicon” such that the plasmid is replicated in a host cell.

[0072] As used herein, “positive control” refers to a “control” that is designed to produce the desired result, provided that all reagents are functioning properly and that the experiment is properly conducted.

[0073] As used herein, “preventative” and “prevent” refers to hindering or stopping a disease or condition before it occurs, even if undiagnosed, or while the disease or condition is still in the sub-clinical phase.

[0074] As used herein, “protein” as used herein refers to a large molecule composed of one or more chains of amino acids in a specific order. The term protein is used interchangeable with “polypeptide.” The order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins are required for the structure, function, and regulation of the body’s cells, tissues, and organs. Each protein has a unique function. The term protein as used herein can also include peptides. Thus, for example, an “effector protein” can include both effector proteins and effector peptides.16U1197.70253WO00#1479354.2

[0075] As used herein, “purified” or “purify” is used in reference to a nucleic acid sequence, peptide, or polypeptide that has increased purity relative to the natural environment.

[0076] As used herein, the term “recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc.). Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

[0077] The terms "treating”, and "treatment" as used herein refer generally to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom, or condition thereof, including but not limited to an inflammatory disease or symptom thereof.

[0078] As used herein, the term “vector” or is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may include a DNA molecule, linear or circular (e.g., plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of both.

[0079] As used herein, “wild-type” is the typical form of an organism, variety, strain, gene, protein, or characteristic as it occurs in nature, as distinguished from mutant or modified forms that may result from selective breeding or transformation with a transgene.

[0080] As used herein, “encoding” can refer to the basic biological concept that DNA can be transcribed into RNA, which then can be translated into a polypeptide.17U1197.70253WO00#1479354.2

[0081] As used herein, the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant IL- 10 is n IL- 10 comprising one or more changes in amino acid residues (i.e., “substitutions”) as compared to a wild type IL- 10 amino acid sequence. The term “variant” encompasses homologous proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a reference sequence (e.g. a wildtype IL- 10 cytokine or fragment thereof) and having the same or substantially the same functional activity or activities as the reference sequence, or having a modified functional activity (e.g. an improved functional activity) relative to the activity of a reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence (e.g. a wildtype IL- 10 cytokine) that display the same or substantially the same functional activity or activities as the reference sequence. The terms “modified” and “variant” in the context of an IL- 10 cytokine are intended to be interchangeable throughout. As such, the terms “modified IL- 10” and “variant IL- 10,” and related terms such as modified IL- 10 cytokines, variant IL- 10 cytokines, modified IL- 10 proteins, or variant IL- 10 proteins, are interchangeable.

[0082] As used herein, the term “IL- 10” or equivalently, “interleukin- 10” or “IL- 10 protein” or “IL- 10 cytokine” and the like are equivalent terms for the purposes of this disclosure and may be used interchangeably. Without being bound by theory, interleukin- 10 (IL- 10) is a multifunctional cytokine with a central role in regulating immune responses. It modulates the behavior of numerous immune cell types and is best known for its strong anti-inflammatory activity. Among its key actions, IL- 10 suppresses the production of pro-inflammatory mediators such as tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and interleukin- 1 (IL-1) in activated macrophages and other antigen-presenting cells. IL- 10 exerts its effects through a heterotetrameric receptor complex composed of two IL-10R1 (also known as IL-lORa) ligandbinding subunits and two IL-10R2 (IL-10RP) accessory subunits required for signaling. Engagement of IL- 10 with the extracellular domain of IL-10R1 triggers activation of the receptor-associated kinases JAK1 (Janus kinase 1) and TYK2 (tyrosine kinase 2). These kinases phosphorylate tyrosine residues within the intracellular tail of IL-10R1 creating docking sites for the transcription factor STAT3 (signal transducer and activator of transcription 3). Upon recruitment, STAT3 is phosphorylated, forms homodimers, and translocates to the nucleus. There, it binds to STAT-responsive promoter elements, driving the expression of IL- 10- regulated genes that collectively down-modulate inflammation and restore immune balance. The wildtype IL- 10 (human) amino acid sequence is represented by SEQ ID NO:1 (wild type18U1197.70253WO00#1479354.2IL- 10)SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 1).

[0083] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.Modified IL-10 Proteins

[0084] Provided here are modified IL- 10 proteins.

[0085] In some embodiments, the modified IL- 10 protein comprises an amino acid sequence selected from any one of the sequences provided in the Sequence Listing, which forms part of this disclosure, including SEQ ID NOs: 2-29 which are variants based on wildtype IL- 10 of SEQ ID NO: 1, as well as an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-29 and which retains at least one amino acid substitution at a position corresponding to D25X, R32X and / or E96X relative to SEQ ID NO: 1, wherein “X” is any amino acid other than the wildtype amino acid.

[0086] In additional embodiments, the modified IL- 10 protein comprises an amino acid sequence selected from any one of the sequences described in the section entitled “SEQUENCES,” including SEQ ID NOs: 2-29 which are variants based on wildtype IL- 10 of SEQ ID NO: 1, as well as an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-29 and which retains at least one amino acid substitution at a position corresponding to D25X, R32X and / or E96X relative to SEQ ID NO: 1, wherein “X” is any amino acid other than the wildtype amino acid.

[0087] In certain embodiments, the modified IL- 10 protein comprises one or more amino acid substitutions at positions corresponding to D25, R32, and / or E96 of SEQ ID NO: 1. Nonlimiting examples include:(a) R32D or R32E substitutions;(b) E96R, E96K, or E96A substitutions;(c) D25A, D25R, or D25K substitutions;19U1197.70253WO00#1479354.2(d) combinations of said substitutions, including but not limited to a modified IL- 10 protein have D25A / D25R / D25K and R32D / R32E substitutions, D25A / D25R / D25K and E96R / E96K / E96A substitutions, and R32D / R32E and E96R / E96K / E96A substitutions;(e) combinations of said substitutions, including but not limited to a modified IL- 10 protein have D25X and R32X paired substitutions, D25X and E96X paired substitutions, and R32X and E96X paired substitutions, wherein “X” is any amino acid substitution other than the wildtype amino acid at the corresponding position in SEQ ID NO: 1;(f) combinations of said substitutions, including but not limited to a modified IL- 10 protein have D25X and R32X and E96X triplet substitutions, wherein “X” is any amino acid substitution other than the wildtype amino acid at the corresponding position in SEQ ID NO: 1; and wherein the modified IL- 10 proteins can maintain normal anti-inflammatory activity while exhibiting no or reduced pro-inflammatory activity compared to wild-type IL- 10.

[0088] In some embodiments wildtype IL- 10 protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, a modified IL- 10 protein is a mutant IL- 10 protein. In some embodiments, the modified IL- 10 protein has amino acid substitution corresponding to D25 of wild-type IL-10 (SEQ ID NO: 1). In some embodiments, the modified IL-10 protein has amino acid substitution corresponding to R32 of wild-type IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has amino acid substitution corresponding to E96 of wild-type IL-10 (SEQ ID NO: 1).

[0089] In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding to D25 and E96 of wild-type IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding to R32 and E96 of wildtype IL- 10 (SEQ ID NO: 1).

[0090] In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding to D25R of wild-type IL-10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding to D25K of wild-type IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL-10 protein has an amino acid substitution corresponding to R32D of wild-type IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding to R32E of wild-type IL-10 (SEQ ID NO: 1). In some embodiments, the modified IL-10 protein has an amino acid substitution corresponding to E96R of wild-type IL- 10 (SEQ ID NO: 1). In some embodiments, 20U1197.70253WO00#1479354.2the modified IL- 10 protein has an amino acid substitution corresponding to E96K of wild-type IL-10 (SEQ ID NO: 1).

[0091] In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding D25K and E96K of wild-type IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding D25K and E96R of wildtype IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding D25R and E96K of wild-type IL-10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding D25R and E96R of wild-type IL- 10 (SEQ ID NO: 1).

[0092] In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding R32D and E96R of wild-type IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding R32E and E96R of wildtype IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding R32E and E96K of wild-type IL- 10 (SEQ ID NO: 1). In some embodiments, the modified IL- 10 protein has an amino acid substitution corresponding R32D and E96K of wild-type IL- 10 (SEQ ID NO: 1).

[0093] In some embodiments, the modified IL- 10 has an amino acid substitution corresponding to D25, R32 and / or E96 of the wild-type IL- 10 protein of SEQ ID NO: 1. In some embodiments, the D25 amino acid substitution is a R or K. In some embodiments, the R32 amino acid substitution is a D or E. In some embodiments, the E96 amino acid substitution is a R or K.

[0094] In some embodiments, the modified IL- 10 has an amino acid sequence of any one of SEQ ID NOs: 2-15. In other embodiments, the modified IL-10 has an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any one of SEQ ID NOs: 2-15. In still other embodiments, the modified IL- 10 has an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any one of SEQ ID NOs: 2-15 and which retains the amino acid substitutions, deletions, or insertions that are present in at least one of SEQ ID NOs: 2-15 relative to the wildtype amino acid of sequence SEQ ID NO: 1. In still other21U1197.70253WO00#1479354.2embodiments, the modified IL- 10 has an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any one of SEQ ID NOs: 2-15 and which retains at least one amino acid substitution, deletion, or insertion selected from the group consisting of that are present in at least one of SEQ ID NOs: 2- 15 relative to the wildtype amino acid of sequence SEQ ID NO: lat least one, at least two, at least three, at least four, at least five, at least six, at least 7.Adeno Associated Virus (AAV) Particles

[0095] The term “AAV” is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all AAV subtypes including both naturally occurring and recombinant forms, unless otherwise indicated.

[0096] ‘ ‘rAAV genomes” comprise a heterologous nucleic acid flanked by 5' and 3' AAV inverted terminal repeats (ITRs). In some embodiments, rAAV genomes may be linear or circular, single-stranded or double- stranded, and / or self-complementary. ITR sequences are about 145 bp in length. While the entire sequences encoding the ITRs are commonly used in engineering rAAVs, modification of these sequences is permissible and may be done using standard techniques (see, e.g., disclosures related to nucleic acid engineering and rAAV particle production in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). For example, artificial ITRs may be engineered for tissue specificity. In some embodiments, a nucleic acid described herein comprises a heterologous nucleic acid (e.g., a nucleic acid encoding a modified IL- 10 protein) flanked by a first AAV ITR and a second AAV ITR.

[0097] The term “AAV particle” or “rAAV particle” refers to a viral particle comprising at least one AAV capsid protein and an encapsidated polynucleotide (e.g., an rAAV genome) which in some embodiments, may be administered to a subject and / or delivered to a selected target cell. Examples of AAV capsid proteins include VP1, VP2, and VP3. As used herein, “target cell” refers to cells that are contacted with rAAV particles (e.g., rAAV particles comprising a heterologous nucleic acid encoding a modified IL-10 protein).

[0098] A “helper vector” comprises a “helper nucleic acid” (e.g., a nucleic acid comprising an El gene, an E2A gene, an E4 gene, and / or a VA gene), which functions in trans for productive AAV replication and encapsidation. In some embodiments, a helper nucleic acid comprises a22U1197.70253WO00#1479354.2plurality of AAV helper genes described herein, such as two, three, four, five, six, seven, eight, nine, ten, or more than ten AAV helper genes. In some embodiments, a helper nucleic acid comprises one or more AAV helper genes (e.g., an El gene, an El gene, an E2A gene, an E4 gene, a VA gene, and / or an LTR gene) derived from an AAV helper virus. Preferably, the AAV helper nucleic acid supports efficient AAV vector production without generating any detectable wild-type AAV particles (e.g., AAV particles containing functional rep and capsid protein genes). Helper nucleic acids, and methods of making said nucleic acids, have been previously described and are commercially available (see, e.g., disclosures related to AAV helper nucleic acids and rAAV particle production in pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini , Journal of Virology, Vol. 79, 5296-5303; Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adeno associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188).

[0099] The “packaging vector” comprises “a packaging nucleic acid” and provides nucleotide sequences (e.g., AAV rep and AAV capsid protein gene sequences) upon which an AAV is dependent for replication (e.g., accessory functions). In some embodiments, a packaging nucleic acid comprises an AAV rep gene sequence and an AAV cap gene sequence.Recombinant AAV Genomes

[0100] In some embodiments, an rAAV particle comprises at least one AAV capsid protein and a nucleic acid (e.g., an rAAV genome) comprising a 5' AAV inverted terminal repeat (ITR), a heterologous nucleic acid, and a 3' AAV ITR. The 5' and 3' AAV ITRs may be alternatively referred to herein as “first” and “second” AAV ITRs, respectively. A “heterologous nucleic acid” refers to any DNA sequence that is not normally found between flanking AAV ITRs. In23U1197.70253WO00#1479354.2some embodiments, rAAV genomes may be linear or circular, single-stranded or doublestranded, and / or self-complementary. In some embodiments, a vector (e.g., a plasmid) comprises an rAAV genome.

[0101] In some embodiments, AAV ITR sequences are about 145 bp in length. While the entire sequences encoding the ITRs are commonly used in engineering rAAVs, modification of these sequences is permissible and may be done using standard techniques (see, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). For example, artificial ITRs may be engineered for tissue specificity.

[0102] In some embodiments, a heterologous nucleic acid is a naturally occurring nucleic acid sequence (e.g., a non-engineered sequence) that is not normally found between AAV ITRs. In some embodiments, a heterologous nucleic acid is an engineered nucleic acid sequence. In some embodiments, a heterologous nucleic acid may contain segments of DNA taken from different organisms. In some embodiments, a heterologous nucleic acid comprises a combination of naturally-occurring and engineered DNA sequences.

[0103] In some embodiments, a heterologous nucleic acid comprises 1-5,000 nucleotides. In some embodiments, heterologous nucleic acids comprise approximately 1-10, 10-20, 20-30, 30- 40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1,000, 1,000-1,100, 1,100-1,200, 1,200-1,300, 1,300-1,400, 1,400-1,500, 1,500-1,600, 1,600-1,700, 1,700-1,800, 1,800-1,900, 1,900-2,000, 2,000-2,100, 2,100-2,200, 2,200-2,300, 2,300-2,400, 2,400-2,500, 2,500-2,600, 2,600-2,700, 2,700-2,800, 2,800-2,900, 2,900-3,000, 3,000-3,100, 3,100-3,200, 3,200-3,300, 3,300-3,400, 3,400-3,500, 3,500-3,600, 3,600-3,700, 3,700-3,800, 3,900-4,000, 4,000-4,100, 4,100-4,200, 4,200-4,300, 4,300-4,400, 4,400-4,500, 4,500-4,600, 4,600-4,700, 4,700-4,800, 4,800-4,900, or 4,900-5,000 nucleotides.

[0104] In some embodiments, a heterologous nucleic acid comprises a sequence that is capable of being expressed in cell (e.g., a transgene). In some embodiments, a heterologous nucleic acid comprises a plurality of sequences wherein at least a first sequence (e.g., a transgene) in the plurality is expressed in a cell and at least a second sequence in the plurality are not expressed in the cell (e.g., repetitive elements, repair templates, etc.). However, in some embodiments, a heterologous nucleic acid is not transcribed into an RNA.24U1197.70253WO00#1479354.2

[0105] In some embodiments, a heterologous nucleic acid encodes a modified IL- 10 protein as disclosed herein. In some embodiments, a heterologous nucleic acid encodes a modified IL- 10 protein of any one of SEQ ID NOs: 2-15.

[0106] In some embodiments, a rAAV comprises a heterologous nucleic acid that encodes a modified IL- 10 protein as disclosed herein. In some embodiments, a rAAV comprises a transgene that encodes a modified IL- 10 protein as disclosed herein. In some embodiments, a rAAV comprises a heterologous nucleic acid comprising a transgene that encodes a modified IL- 10 protein as disclosed herein. In some embodiments, a rAAV comprises a heterologous nucleic acid that encodes a modified IL- 10 protein as disclosed herein. In some embodiments, a rAAV comprises a transgene that encodes a modified IL- 10 protein as disclosed herein. In some embodiments, a rAAV comprises a heterologous nucleic acid comprising a transgene that encodes a modified IL-10 protein of any one of SEQ ID NOs: 2-15.

[0107] In some embodiments, a heterologous nucleic acid comprises at least one transgene. As used herein, “transgene” refers to a DNA sequence which encodes an RNA to be expressed in a cell. In some embodiments, a heterologous nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more transgenes. In some embodiments, a transgene comprises a cDNA sequence. In some embodiments, RNA expressed from a transgene is not translated into a protein. In some embodiments, a transgene encodes an mRNA that is translated into a protein. In some embodiments, a transgene may comprise multiple DNA sequences (e.g., encodes multiple RNA species).

[0108] Generally, a heterologous nucleic acid (e.g., one comprising one or more transgenes) is designed to comprise a size that is within the packaging capacity of AAVs. In some embodiments, a heterologous nucleic acid (e.g., one comprising one or more transgenes) comprises a sequence that is 5kb or less, such as about 4.8kb, 4.7kb, 4.6kb, 4.5kb, or less. Nonlimiting examples of sequences found in transgenes include sequences encoding small-hairpin RNAs (shRNAs), short-interfering RNAs (siRNAs), prokaryotic -interfering RNAs (prosiRNAs), micro-RNAs (miRNAs), long non-coding RNAs (IncRNAs), Piwi-interacting RNAs (piRNAs), exon-skipping RNAs, enzymatic RNAs, guide RNAs ((gRNAs), e.g., single-guide RNAs (sgRNAs)), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and messenger RNAs (mRNAs).25U1197.70253WO00#1479354.2

[0109] In some embodiments, a transgene encodes a modified IL- 10 protein as disclosed herein. In some embodiments, a transgene encodes a modified IL- 10 protein of any one of SEQ ID NOs: 2-15.Regulatory Sequences

[0110] The term “regulatory sequence” may be used herein to refer to a nucleic acid sequence that is capable of modulating the expression, stability, and / or levels of an RNA (e.g., an mRNA) and / or a peptide or protein product thereof when operably linked to a gene sequence encoding the RNA. A nucleic acid sequence (e.g., a sequence comprising a gene) and a regulatory sequence may be referred to as “operably linked” when they are associated in such a way (e.g., via a covalent bond) as to place the expression of the nucleic acid sequence under the influence or control of the regulatory sequence. As a non-limiting example, a gene sequence may be referred to as operably linked to a promoter if induction of the promoter results in the transcription of a coding sequence comprised in the gene, if the nature of the linkage between the gene and the promoter does not result in the introduction of a frame- shift mutation, and / or interfere with the ability of the promoter to direct the transcription of the coding sequence.

[0111] Non-limiting examples of regulatory sequences include a promoter, an enhancer, a silencer, a transcription factor binding sequence, a 5’ UTR, a 3’ UTR, a translation initiation regulatory sequence, a transcriptional start sequence, a transcription termination sequence, an acceptor / donor splicing site, a mRNA degradation or decay signal, a polyadenylation signal, a translation initiation codon, a RNA-binding protein binding site, a ribosome binding site, a ribozyme, an intron, a translation termination sequence, and a stop codon. In some embodiments, a regulatory sequence is a transcriptional regulatory sequence (e.g., a promoter), a post-transcriptional regulatory sequence (e.g., a splicing regulatory signal or a polyadenylation poly(A) signal), or a translation regulatory sequence (e.g., a translation initiation or termination regulatory sequence).

[0112] In some embodiments, a heterologous nucleic acid comprises at least one regulatory sequence described herein. In some embodiments, a regulatory sequence is native to a nucleic acid or a gene comprised therein. In some embodiments, a regulatory sequence is heterologous to a nucleic acid or a gene comprised therein. In some embodiments, a heterologous nucleic acid comprises a gene (e.g., a heterologous nucleic comprising a transgene) which is operably linked to at least one regulatory sequence. In some embodiments, a heterologous nucleic acid comprises a plurality of regulatory sequences, such as one comprising a gene that is operably26U1197.70253WO00#1479354.2linked to a plurality of regulatory sequences. However, in some embodiments, a heterologous nucleic acid comprises a regulatory sequence that is not operably linked to a gene sequence encoding an RNA.

[0113] A promoter, generally, is a region of nucleic acid that initiates transcription of a nucleic acid encoding a product.

[0114] To achieve exogenous expression levels of modified IL- 10, any of a number of promoters may be employed. The promoter may be, for example, a constitutive promoter, tissue-specific promoter, inducible promoter, or a synthetic promoter.

[0115] In some embodiments, a promoter is a constitutive promoter (e.g., Herpes Simplex virus (HSV) promoter, the thymidine kinase (TK) promoter, the Simian Virus 40 (SV40) promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, the CAG promoter, the human elongation factor- 1 alpha (EFla) promoter, an RNA pol II promoter, an RNA pol III promoter, a U6 promoter, a Hl promoter, an RNA pol II promoter, a chicken P-actin (CB or CBA) promoter, etc.), an inducible promoter (e.g., cytochrome P450 gene promoters, heat shock protein gene promoters, metallothionein gene promoters, hormone-inducible gene promoters, such as the estrogen gene promoter zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex) -inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible, the rapamycin-inducible system, etc.), or a tissue-specific promoter (e.g., a neuron- specific promoter, a skeletal muscle-specific promoter, a smooth muscle- specific promoter, a cardiac muscle- specific promoter, etc.). In some embodiments, the promoter is a chicken P-actin (CB or CBA) promoter rAAV for delivery of modified IL-10 protein

[0116] As a vehicle for delivering heterologous nucleic acid comprising a transgene encoding a modified IL- 10 encoding, recombinant adeno-associated (rAAV) particles are used. AAV has emerged as one of the most favorable vehicles for gene therapy in clinical applications. Its benefits are related to its increased safety, the low immunogenic profile of transduced cells, and its ability to provide prolonged, effective transgene expression in joint tissues. Recent advances in AAV technology, including the development of self-complementary (double- stranded)27U1197.70253WO00#1479354.2vectors, and improved methods for high-titer vector production, have further increased its potential for mainstream clinical use.

[0117] An AAV particle disclosed herein may be of any AAV serotype (e.g., AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13), including any derivative (including non-naturally occurring variants of a serotype) or pseudotype. Non-limiting examples of derivatives and pseudotypes include AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives / pseudotypes, and methods of producing such derivatives / pseudotypes are known in the art (see, e.g., Mol. Ther. 2012 Apr; 20(4):699-708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan A, Schaffer DV, Samulski RJ.). In some embodiments, the AAV particle is a pseudotyped AAV particle, which comprises a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2 or AAV3) and a capsid comprised of capsid proteins derived from another serotype (i.e., a serotype other than AAV2 or AAV3, respectively). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001)).

[0118] In some embodiments, a rAAV particle is self-complimentary, in that it contains a region of the nucleic acid that is complementary to another region of the nucleic acid, initiating the formation of the double-strandedness of the nucleic acid comprised in a particle.

[0119] In some embodiments, an AAV particle disclosed herein is a recombinant AAV (rAAV) particle, e.g., comprising a nucleic acid sequence or transgene. In some embodiments, the nucleic acid sequence encodes a modified IL- 10 protein as disclosed herein. In some embodiments, the transgene encodes a modified IL- 10 protein as described herein.Methods of packaging rAAV particles

[0120] Non-limiting methods of producing rAAV particles that comprise a transgene encoding a modified IL- 10 protein are described herein. Other methods are also known in the art and commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by 28U1197.70253WO00#1479354.2reference; Li et al., J. Virol, 2012, v.86(15)and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a transgene encoding a modified IL- 10 protein may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), and transfected into recombinant cells such that the rAAV particle can be packaged and subsequently purified.

[0121] In some embodiments, packaging is performed in a helper cell or producer cell, such as a mammalian cell or an insect cell. Non-limiting examples of mammalian cells include, but are not limited to, HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (see, e.g., ATCC® CRL-1573™, ATCC® CRL-1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-10™, or ATCC® CCL-61™). Exemplary insect cells include, but are not limited to Sf9 cells (see, e.g., ATCC® CRL-1711™). The helper cell may comprise rep and / or cap genes that encode the Rep protein and / or Cap proteins for use in a method described herein. In some embodiments, the packaging is performed in vitro.

[0122] In some embodiments, a plasmid encoding a modified IL- 10 protein is combined with one or more helper plasmids, e.g., that contain a rep gene of a first serotype and a cap gene of the same serotype or a different serotype, and transfected into helper cells such that the rAAV particle is packaged.

[0123] In some embodiments, the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene, and a second helper plasmid comprising one or more of the following helper genes: Ela gene, Elb gene, E4 gene, E2a gene, and VA gene. For clarity, helper genes are genes that encode helper proteins Ela, Elb, E4, E2a, and VA. In some embodiments, the rep gene is a rep gene derived from AAV3, AAV5, or AAV6 and the cap gene is derived from AAV2, AAV3, AAV5, or AAV6 and may include modifications to the gene in order to produce the modified capsid protein described herein. In some embodiments, the cap gene is modified such that one or more of the proteins VP1, VP2 and VP3 do not get expressed. In some embodiments, the cap gene is modified such that VP2 does not get expressed. Methods for making such modifications are known in the art (Lux et al. (2005), J Virology, 79: 11776-87)

[0124] Helper plasmids, and methods of making such plasmids, are known in the art and commercially available (see, e.g., pDF6, pRep, pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA;29U1197.70253WO00#1479354.2Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188). Plasmids that encode wild-type AAV coding regions for specific serotypes are also known and available. For example, pSub201 is a plasmid that comprises the coding regions of the wild-type AAV2 genome (Samulski et al. (1987), J Virology ,6:3096-3101).

[0125] An exemplary, non-limiting, rAAV particle production method is described next. One or more helper plasmids are produced or obtained, which comprise rep and cap ORFs for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some embodiments, the one or more helper plasmids comprise rep genes for a first serotype (e.g., AAV3, AAV5, and AAV6), cap genes (which may or may not be of the first serotype) and optionally one or more of the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some embodiments, the one or more helper plasmids comprise cap ORFs (and optionally rep ORFs) for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The cap ORF may also comprise one or more modifications to produce a modified capsid protein as described herein. HEK293 cells (available from ATCC®) are transfected via CaPO4-mediated transfection, lipids or polymeric molecules such as Polyethylenimine (PEI) with the helper plasmid(s) and a plasmid containing a nucleic acid vector described herein. The HEK293 cells are then incubated for at least 60 hours to allow for rAAV particle production. Alternatively, the HEK293 cells are transfected via methods described above with AAV-ITR containing any one of the recombinant nucleic acids described herein, a helper plasmid comprising genes encoding Rep and Cap proteins, and coinfected with a helper virus. Helper viruses are viruses that allow the replication of AAV.Examples of helper virus are adenovirus and herpesvirus.30U1197.70253WO00#1479354.2

[0126] Alternatively, in another example Sf9-based producer stable cell lines are infected with a single recombinant baculovirus containing any one of the recombinant nucleic acids provided herein. As a further alternative, in another example HEK293 or BHK cell lines are infected with a HSV containing the nucleic acid vector and optionally one or more helper HSVs containing rep and cap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to allow for rAAV particle production. The rAAV particles can then be purified using any method known in the art or described herein, e.g., by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.Pharmaceutical compositions

[0127] Provided herein is a pharmaceutical composition comprising any one of the modified IL- 10 proteins or rAAV particles disclosed herein. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier that aids in the delivery of the modified IL-10 proteins. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier that aids in the delivery of the rAAV particles comprising a nucleic acid encoding a modified IL- 10 protein to a subject. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the rAAV particle is administered.

[0128] Pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions (e.g., sterilized, pyrogen-free saline) and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. USP grade carriers and excipients are particularly useful for delivery of rAAV particles to mammalian subjects. Such compositions may further optionally comprise a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof. Methods for making such compositions are well known and can be found in, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2012.

[0129] The pharmaceutical forms of the modified IL- 10 compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the form is sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. In some embodiments, the form is sterile.31U1197.70253WO00#1479354.2The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0130] The pharmaceutical forms of the rAAV particle compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the form is sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. In some embodiments, the form is sterile. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0131] For administration of an injectable aqueous solution, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570- 1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for mammalian administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards generally accepted in veterinary sciences.

[0132] Typically, such compositions may contain at least about 0.1% of the therapeutic agent (e.g., modified IL- 10 protein or rAAV particle) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of therapeutic agent(s) (e.g., rAAV particle) in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of32U1197.70253WO00#1479354.2administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.Routes of Administration

[0133] In some embodiments, the pharmaceutical formulations described herein may be in a dosage form. The dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, epidural, intracranial, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavemous, intrathecal, intravireal, intracerebral, and intracerebroventricular and intradermal. Such formulations may be prepared by any method known in the art.

[0134] Dosage forms adapted for oral administration can be discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or nonaqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, the pharmaceutical formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the pharmaceutical formulation. Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as foam, spray, or liquid solution. In some embodiments, the oral dosage form can contain about 1 ng to 1000 g of a pharmaceutical formulation containing a therapeutically effective amount or an appropriate fraction thereof of the targeted effector fusion protein or composition containing the targeted effector fusion protein The oral dosage form can be administered to a subject in need thereof.

[0135] Where appropriate, the dosage forms described herein can be microencapsulated. The dosage form can also be prepared to prolong or sustain the release of any ingredient. In some embodiments, the targeted effector fusion protein is the ingredient whose release is delayed. In other embodiments, the release of an optionally included auxiliary ingredient is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams &33U1197.70253WO00#1479354.2Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and delayed release dosage forms of tablets and pellets, capsules, and granules. The delayed release can be anywhere from about an hour to about 3 months or more.

[0136] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0137] Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and / or pH dependent polymers, with or without water insoluble / water soluble non polymeric excipient, to produce the desired release profile. The coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, “ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form.

[0138] Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treatments of the eye or other external tissues, for example the mouth or the skin, the pharmaceutical formulations are applied as a topical ointment or cream. When formulated in an ointment, the targeted effector fusion protein, auxiliary active ingredient, and / or pharmaceutically acceptable salt thereof can be formulated with a paraffinic or water- miscible ointment base. In other embodiments, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.

[0139] Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, the targeted effector fusion protein, the composition containing a targeted effector fusion protein, auxiliary active ingredient, and / or pharmaceutically acceptable salt thereof in a dosage form adapted for inhalation is in a particle- size-reduced form that is obtained or obtainable by micronization. In some34U1197.70253WO00#1479354.2embodiments, the particle size of the size reduced (e.g., micronized) compound or salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions / suspensions of an active ingredient, which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators.

[0140] In some embodiments, the dosage forms are aerosol formulations suitable for administration by inhalation. In some of these embodiments, the aerosol formulation contains a solution or fine suspension of the targeted effector fusion protein, the composition containing a targeted effector fusion protein, and / or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be presented in single or multi-dose quantities in sterile form in a sealed container. For some of these embodiments, the sealed container is a single dose or multi-dose nasal or an aerosol dispenser fitted with a metering valve (e.g., metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.

[0141] Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon. The aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer. The pressurized aerosol formulation can also contain a solution or a suspension of a targeted effector fusion protein, composition containing a targeted effector fusion protein, or a pharmaceutical formulation thereof. In further embodiments, the aerosol formulation also contains co-solvents and / or modifiers incorporated to improve, for example, the stability and / or taste and / or fine particle mass characteristics (amount and / or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2, or 3 doses are delivered each time.

[0142] For some dosage forms suitable and / or adapted for inhaled administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to the targeted effector fusion protein, the composition containing a targeted effector fusion protein, an auxiliary active ingredient, and / or pharmaceutically acceptable salt thereof, such a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and / or starch. In some35U1197.70253WO00#1479354.2of these embodiments, the targeted effector fusion protein, the composition containing a targeted effector fusion protein, auxiliary active ingredient, and / or pharmaceutically acceptable salt thereof is in a particle-size reduced form. In further embodiments, a performance modifier, such as L-leucine or another amino acid, cellobiose octaacetate, and / or metals salts of stearic acid, such as magnesium or calcium stearate.

[0143] In some embodiments, the aerosol formulations are arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the targeted effector fusion proteins or compositions containing the targeted effector fusion protein described herein.

[0144] Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas.

[0145] Dosage forms adapted for parenteral administration and / or adapted for any type of injection (e.g. intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular) can include aqueous and / or non-aqueous sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. The dosage forms adapted for parenteral administration can be presented in a single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The doses can be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration.Extemporaneous injection solutions and suspensions can be prepared in some embodiments, from sterile powders, granules, and tablets.

[0146] Dosage forms adapted for ocular administration can include aqueous and / or nonaqueous sterile solutions that can optionally be adapted for injection, and which can optionally contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the eye or fluid contained therein or around the eye of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.

[0147] For some embodiments, the dosage form contains a predetermined amount of the targeted effector fusion protein or composition containing a targeted effector fusion protein per unit dose. In an embodiment, the predetermined amount of the targeted effector fusion protein or 36U1197.70253WO00#1479354.2composition containing a targeted effector fusion protein is a therapeutically effective amount of the targeted effector fusion protein or composition containing a targeted effector fusion protein to treat or prevent an enzymatic deficiency, an immune dysfunction disease, and / or a symptom thereof. In other embodiments, the predetermined amount of the targeted effector fusion protein or composition containing a targeted effector fusion protein can be an appropriate fraction of the therapeutically effective amount of the active ingredient. Such unit doses may therefore be administered once or more than once a day. Such pharmaceutical formulations may be prepared by any of the methods well known in the art.Subjects

[0148] Aspects of the disclosure relate to methods for use with a subject, such as human or non-human animal subjects; with a host cell in situ in a subject; or with a host cell derived from a subject (e.g., ex vivo or in vitro).

[0149] In some embodiments, a subject can be a human (e.g., a male or female of any age group, such as a pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In some embodiments, the non- human animal is a mammal (e.g., primate, such as cynomolgus monkey or rhesus monkey). Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus or rhesus macaques), marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. In some embodiments, the non-human subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject has, is suspected of having, is at risk of having, or is expected to develop a disease, disorder, or condition.

[0150] In some embodiments, the subject has, is suspected of having, or at risk of developing a disease, disorder, or condition. In some embodiments, the disease, disorder, or condition comprises a genetic disease, cancer, inflammatory disease or an inflammatory disorder, autoimmune disease, liver disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, a kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or other disease, or any combination thereof. In some embodiments, the disorder is an inflammatory disorder. In some embodiments, the disorder is an autoimmune disorder. In37U1197.70253WO00#1479354.2some embodiments, the disorder is selected from colitis, sepsis, inflammatory bowel syndrome (IBD), rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), Grave’s disease, Sjorgren’s syndrome, Multiple Sclerosis, allergic asthma, asthma, or autoimmune lymphoproliferative syndrome.

[0151] In some embodiments, the subject has one or more diseases, disorders, or conditions. In some embodiments, the disease, disorder, or condition comprises a genetic disease, cancer, inflammatory disease or an inflammatory disorder, autoimmune disease, liver disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, a kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or other disease, or any combination thereof. In some embodiments, the disorder is an inflammatory disorder. In some embodiments, the disorder is an autoimmune disorder. In some embodiments, the disorders are selected from colitis, sepsis, inflammatory bowel syndrome (IBD), rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), Grave’s disease, Sjorgren’s syndrome, Multiple Sclerosis, allergic asthma, asthma, or autoimmune lymphoproliferative syndrome.Methods of Administration

[0152] In some embodiments, a method comprises administering a modified IL- 10 protein as described herein to a subject in need thereof.

[0153] In some embodiments, a method comprises administering an rAAV particle described herein to a subject in need thereof. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of at least 1 x 105vector genomes (vg) per kilogram (kg) (vg / kg) of a subject’s body weight. In some embodiments, the rAAV particle is administered in a composition (e.g., a pharmaceutical composition) comprising a dose described herein. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 106vg / kg, 2 x 106vg / kg, 3 x 106vg / kg, 4 x 106vg / kg, 5 x 106vg / kg, 6 x 106vg / kg, 7 x 106vg / kg, 8 x 106vg / kg, 9 x 106vg / kg, 1 x 107vg / kg, 2 x 107vg / kg, 3 x 107vg / kg, 4 x 107vg / kg, 5 x 107vg / kg, 6 x 107vg / kg, 7 x 107vg / kg, 8 x 107vg / kg, 9 x 107vg / kg, 1 x 108vg / kg, 2 x 108vg / kg, 3 x 108vg / kg, 4 x 108vg / kg, 5 x 108vg / kg, 6 x 108vg / kg, 7 x 108vg / kg, 8 x 108vg / kg, 9 x 108, 1 x 109vg / kg, 2 x 109vg / kg, 3 x 109vg / kg, 4 x 109vg / kg, 5 x 109vg / kg, 6 x 109vg / kg, 7 x 109vg / kg, 8 x 109vg / kg, 9 x 109vg / kg, vg / kg, 1 x IO10vg / kg, 2 x IO10vg / kg, 3 x IO10vg / kg, 4 x IO10vg / kg, 5 x IO10vg / kg, 6 x IO10vg / kg, 7 x IO10vg / kg, 8 x38U1197.70253WO00#1479354.2IO10vg / kg, 9 x IO10vg / kg, or more. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 106vg / kg to 1 x 1015vg / kg. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 107vg / kg to 1 x 1014vg / kg. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 108vg / kg to 1 x 1013vg / kg. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 109vg / kg to 1 x 1012vg / kg.

[0154] An amount of the modified IL- 10 protein, rAAV, compositions, and pharmaceutical formulations thereof described herein can be administered to a subject in need thereof one or more times per day, week, month, or year. In some embodiments, the amount administered can be the therapeutically effective amount of the targeted effector fusion protein, compositions, and pharmaceutical formulations thereof. For example, the targeted effector fusion protein, compositions, and pharmaceutical formulations thereof can be administered in a daily dose. This amount may be given in a single dose per day. In other embodiments, the daily dose may be administered over multiple doses per day, in which each containing a fraction of the total daily dose to be administered (sub-doses). In some embodiments, the amount of doses delivered per day is 2, 3, 4, 5, or 6. In further embodiments, the compounds, formulations, or salts thereof are administered one or more times per week, such as 1, 2, 3, 4, 5, or 6 times per week. In other embodiments, the targeted effector fusion protein, compositions, and pharmaceutical formulations thereof can be administered one or more times per month, such as 1 to 5 times per month. In still further embodiments, the targeted effector fusion protein, compositions, and pharmaceutical formulations thereof can be administered one or more times per year, such as 1 to 11 times per year.EXAMPLESExample 1. Computationally designed 11-10 mutants

[0155] IL- 10 has both inflammatory and proinflammatory functions, which makes it challenging to use IL- 10 as a therapeutic drug. Based on structural information of IL10 / IL- 10Ra / IL10Rp, the strategy was to mutate IL-10 residues that interact with the DI and D2 domains of IL-10RP to reduce the proinflammatory effect.

[0156] Based on structural information of a IL10 / IL-10Ra / IL10RP complex, IL- 10 residues that interact with the DI and D2 domains of IL-10RP were mutated. With computational39U1197.70253WO00#1479354.2modeling of the interaction between human IL- 10 and IL-10R P-subunit, two candidate reaction sites (designated as A and B) were identified. Compared to the wildtype (WT), mutant A (hlL- 10A - corresponding to SEQ ID NO: 2) and mutant B (hIL-lOB - corresponding to SEQ ID NO: 3), are weaker in binding to IL-10RP; the MMPBSA enthalpy are 8.0Kcal / mol and 12.9Kcal / mol weaker, respectively. The resulting three mutant IL- 10 molecules are hIL-lOA (R32D) (corresponding to SEQ ID NO: 2), hIL-10B(E96R) (corresponding to SEQ ID NO: 3), and hIL-lOC (R32D-E96R) (corresponding to SEQ ID NO: 4). FIG. 1 shows a map of the vector constructs that express wild type (rAAV-CB-hlLlOW or “W”) or mutant human IL- 10 (mutant rAAV-CB -hIL-lOA or “A”; mutant rAAV-CB-hIL-lOB or “B”; mutant rAAV-CB-hlL- 10C or “C”).Example 2. Transfection of HEK cells with mutant IL-10

[0157] Vector constructs were generated for wild type hIL-10 (W), mutant hIL-lOA (A), mutant hIL-lOB (B), and mutant hIL-lOC (C) (FIG. 1). For vector construction, two unique restriction digestion sites (Hindlll at 5 ’-end and Notl at 3 ’-end) for all 4 genes were generated. A unique restriction digestion site for each mutant was inserted to identify each mutant IL- 10.

[0158] HEK293 cells were transfected with the 4 vector plasmids. After transfection, the hlL- 10W and hIL-10 mutants (A, B and C) were detected via hIL-10 ELISA. High levels of hIL-10 and hIL-10 mutants (W, A, B and C) were detected in cell culture medium for each construct.Example 3. Function of WT and mutant IL- 10 in peripheral blood mononuclear cells (PBMCs)

[0159] To test the function of wild type and mutant hIL-10 (W, A, B and C), PBMCs were treated with the WT hIL-10 (W), hIL-lOA (A), hIL-lOB (B), and hIL-lOC (C) proteins expressed by the HEK293 cells of Example 2. PBMCs were treated with the hIL-10 proteins for 20 minutes with a high dose (20nM) or low dose.(lOnM). The high dose was 2 folds higher than the low dose. Total cellular proteins were then subjected to western blot analysis.

[0160] As phosphorylation of STAT3 (pSTAT3) occurs during IL- 10 signaling, pSTAT3 was measured, as an indicator of IL- 10 activation. WT hIL-10 stimulated pSTAT3 levels in dosedependent manner, while all mutants of hIL-10 have much lower activities indicating the mutations altered the receptor bindings (FIG. 2).40U1197.70253WO00#1479354.2Example 4. In vitro evaluations of IL-10 and IL-10 mutants in pSTAT3 responsive cell lines

[0161] To test the function of wildtype hIL-10 (W) and hIL-10 mutants (A, B and C), hIL-10 was measured in the cell culture of the transfected HEK293 cell of Example 2. The levels of hIL-10 (W, A, B and C) in the culture medium was quantified by human IL- 10 ELISA. The cell culture media containing wildtype hIL-10 (W) and mutant hIL-10 (A, B and C) were then used to treat THP-Luc and U937-Luc cells (human monocytes transduced with lenti-pSTAT3 responsive promoter- luciferase gene). As shown in FIGs. 3 A and 3B, the IL- 10 mutant C has reduced activity compared with wild type IL- 10 (W) in both cell types, while mutant A and B have nearly no activity. To test if human IL- 10 (W) and IL10 mutants (A, B and C) are functional in mouse cells, the media containing wildtype hIL-10 (W) and mutant hIL-10 (A, B and C) were used to treat RAW-Luc cells (mouse macrophage cell derived). As shown in Fig 3C, W, B, and C are clearly functional although A has no detectable activity. These data also confirms that IL- 10 mutant B is functional although its activity was not observed in THP-Luc cells and U937-Luc cells.

[0162] In addition, mutant mouse IL- 10 (X, Y, and Z) was generated in the same method as the human IL- 10. Mutant mlL-lOX (X) corresponds to the same IL- 10 mutation as hIL-lOA (A), mutant mlL-lOY (Y) corresponds to the same IL- 10 mutation as hIL-lOB (B), and mutant mlL- 10Z (Z) corresponds to the same IL- 10 mutation as hIL-lOC (C). WT mouse IL- 10 (W) and the mutant IL- 10 (X, Y, and Z) were tested for their activities in vitro using RAW-Luc cells. As shown in Fig 3D, mlL-lOX and mlL-lOY have reduced activities when compared to wild type mlL-lOW.Example 5. In vivo evaluation of wildtype and mutant IL- 10 transfection in a mouse model of colitis

[0163] To test the effect of the mutant hIL-10 protein on disease severity, female C57BL / 6 mice were purchased from Jackson Laboratory at 8 weeks of age. After a few days of acclimation, animals were randomly assigned in to 7 groups: Ctrl-control group (n=9) received PBS injection as a normal control; MD Ctrl (n=10)-received PBS and DSS-treatment serve as a disease model control. GFP, W, A, B and C groups (n=10) received rAAV vectors expressing GFP, W, A, B and C, respectively. The rAAV8 vectors (1x1011 vg / mouse) were injected interperitoneally (IP) in GFP, W, A, B and C group. Nineteen days after vector injection, dextran sodium sulfate (DSS) (4% in drinking water, 5 days) induction was performed. The body weight (BW) and disease development were assessed daily.41U1197.70253WO00#1479354.2

[0164] FIG. 4 shows representative data of transgene hIL-10 (W, A, B and C) expression detected in mouse serum by hIL-10 specific ELISA. These data indicate that the rAAV8 vectors are functional and mediated high levels of transgene expression in the mouse model. Detected levels of hIL-10 (W, A, B and C) were observed to be decreased after DSS treatment indicating the possibility that the disease induction (or development) may increase the consumption (or demand) of IL- 10.

[0165] FIG. 5 shows that the disease activity index (DAI) of each group 7 days after DSS- induction. Group C had a significantly lower DAI score in comparison to the W and B groups. These differences were not observed in later days as the animals were recovered, which is commonly seen in this acute IBD model (data not shown).

[0166] 14 days after DSS-induction the experiment was terminated, and pathological analysis was performed. FIG. 6 shows that the spleen weights at 14 days post DSS-induction in group C, were significantly lower than that of the W, A, and B groups. Spleen weight is one hallmark of inflammation, indicating that high dose hIL-10 has a proinflammatory effect and the mutant hIL-10C has a significantly lower proinflammatory effect.

[0167] Next, splenocytes from each animal were isolated and subjected to flowcytometry analyses using pSTAT3 levels as an indicator for the response of treatments. As shown in FIGs. 7A-7C, the IL- 10 mutants (A, B and C) have similar effects on monocytes (FIG.7C), but significantly reduced effects on NK cells and T cells (FIGs. 7A and 7B).Example 6. IL- 10 Gene Therapy in IL- 10 knockout mouse model of colitis

[0168] Next, IL- 10 knockout (IL- 10 KO) mice were used to evaluate the therapeutic effect of the mutant IL- 10 proteins. IL- 10 knockout (IL- 10 KO) mice spontaneously develop unremitting colitis; however, the phenotype highly depends on the genetic background. Recently developed IL- 10 KO mice on Balb / c background (BALB / cAnNTac-I110em7Tac) can develop high incidence by 24 weeks of age, thus providing a reliable and faithful mouse model for the therapeutic evaluations of candidate drugs (Covington et al, Eur. J. Pharmacol.2020;885: 173505).

[0169] Cohorts of IL- 10 KO mice (male, 8 weeks of age from Taconic, USA) were IP injected with rAAV8 vectors expressing IL- 10 (W) or the IL- 10 mutants (A, B and C), GFP (lOel 1 vg / mouse), or PBS (as a model control or NC as no vector injected control). An additional group of Balb / c mice that received PBS were used as a normal control (Bal). FIG. 8 showed that the vectors mediated similarly high levels of transgene IL- 10 expression. FIGs. 9A-9E showed that 42U1197.70253WO00#1479354.2the body weight increases in W, A, B, and C groups were significantly higher than in GFP (the vector control) group. Importantly, the DAI scores in W, A, B and C groups were significantly lower than GFP group and are similar to Balb / c (normal control group, FIGs. 10A-10E).Example 7. Functional evaluations of computationally designed IL- 10 mutants and gene delivery in IBP mouse models

[0170] Interleukin- 10 (IL- 10) is immunoregulatory cytokine that can mediate both antiinflammatory and pro-inflammatory effects. IBD is a chronic inflammatory disease that can cause severe scarring and inflammation in the GI tract. In some embodiments, a IL- 10 mutant drug that has anti-inflammatory but no pro-inflammatory effect for the treatment of IBD was developed. Using structure based computational approach, several IL- 10 mutants were created which generated AAV vectors for gene therapy. In vitro functional testing was done in generated cell lines (THP-luc, U-937-luc and RAW-luc) via luciferase assay, and some of the mutants were functional. Two different mouse models (DSS induced and IL-10KO) were used to test the therapeutic and protective effect of the IL- 10 mutants. As described in some embodiments, the mutations of IL- 10 altered its functions, and gene delivery enhanced the therapeutic potential for chronic diseases such as IBD. The data and results are shown in FIGs. 11-15 and described below and in the accompanying Brief Description of the Drawings.

[0171] FIG.l 1 provides an overview of the strategy used to reduce the proinflammatory effect of IL- 10 by computational design of IL- 10 mutants.

[0172] FIGs. 12A-12C describe how the IL- 10 plasmid construct was transfected into HEK 293 cells and then the media was collected and used for luciferase assay testing on cell lines (FIG. 12A). The IL-10 media was tested at various concentrations (0, 5, 15 and 30 ng / ml) to show the IL- 10 expression. THP-1 are a human monocytes cell line that showed IL- 10 expression from the media (FIG. 12B). RAW-263 cells are mouse monocyte that showed IL-10 expression from the media doses (FIG. 12C).

[0173] FIGs. 13A-13E provide an overview of the model used. The overview of this model was to determine the protective effect and therapeutic potential of IL- 10 mutants as shown in Fig. 13A. Mice were injected with rAAV8 virus expressing IL-10 mutant 14-16 days before they drank 4% DSS water for 5 days. After 10 days, mice were sacrificed to be analyzed via flow cytometry and histology. IL- 10 Elisa was done with serum samples that were collected over the duration of the study (FIG. 13B). The ELISA indicated continued IL-10 expression from the rAAV8 virus. The model was scored using the study (FIG. 13C). The spleen weight was43U1197.70253WO00#1479354.2analyzed to indicate the how much inflammation was present due to IBD (FIG. 13D). Flow cytometry was conducted on mouse spleenocytes to look at pSTAT3 expression in T-cells, NK cells and Monocytes via Mean Fluorescence Intensity(MFI) (FIG. 13E).

[0174] FIG s. 14A-14E provide an overview of this model in IL-IOKO mice. Thismodel was used to determine the therapeutic effect of the IL- 10 mutants in IL-IOKO mice (FIG 14A). An IL- 10 ELISA was done with the mouse serum to demonstrate the continued expression from the rAAV8 virus at Weeks 2 or 4 (FIG. 14B). The DAI scoring and body weight was analyzed by AUC to show the overall progression of IBD in each of the groups. The histology, spleen weight and colon weight were conducted to demonstrate the potential of the IL- 10 mutants (FIG. 14C). In addition, the average of colon thickness in each group was determined (FIG. 14D).The representative images of H&E stained colon sections from each group. Balb / c mice (n=4) as a normal mice control. Saline, (n=5) IL- 10 KO mice injected with saline as a disease model control. GFP, (n=4) IL- 10 KO mice injected with rAAV8-GFP as a vector control. W (n=4), A (n=5), B (n=5) and C (n=5), IL- 10 KO mice injected with rAAV vectors expressing W, A, B and C, respectively. *, p<0.05, **, P<0.01, ***, p<0.001. (FIG. 14E).

[0175] FIGs. 15A-15E show the effects of IL-10 (W, A, B, and C constructs) on pSTAT3 levels in immune cells in the IL-IOKO mice. (FIG. 15A) on CD4 T cells. (FIG. 15B) on T effector CD8 cells. (FIG. 15C) on NK cells. (FIG. 15D) on monocytes. (FIG. 15E) on Macrophages. Saline, (n=5) IL-10 KO mice injected with saline as a disease model control. GFP, (n=4) IL- 10 KO mice injected with rAAV8-GFP as a vector control. W (n=4), A (n=5), B (n=5) and C (n=5), IL- 10 KO mice injected with rAAV vectors expressing W, A, B and C, respectively. A vs B, p<0.05.SEQUENCES

[0176] In various embodiments, the present disclosure provides variant IL- 10 cytokines which are derived from the wildtype IL- 10 cytokine comprising SEQ ID NO: 1. SEQ ID NO: 1 is as follows:

[0177] SEQ ID NO: 1 (wild type IL- 10)SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN44U1197.70253WO00#1479354.2

[0178] In various embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15.

[0179] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains at least one of the following substitutions: D25X, R32X, or E96X, wherein “X” is any amino acid other than the wildtype amino acid at that position relative to SEQ ID NO: 1.

[0180] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains at least two of the following substitutions: D25X, R32X, or E96X, wherein “X” is any amino acid other than the wildtype amino acid at that position relative to SEQ ID NO: 1.

[0181] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains all three of the following substitutions: D25X, R32X, or E96X, wherein “X” is any amino acid other than the wildtype amino acid at that position relative to SEQ ID NO: 1.

[0182] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains at least one of the following substitutions: D25X, R32X, or E96X, wherein “X” is any conservative amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1.

[0183] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 45U1197.70253WO00#1479354.297%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains at least two of the following substitutions: D25X, R32X, or E96X, wherein “X” is any conservative amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1.

[0184] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains all three of the following substitutions: D25X, R32X, or E96X, wherein “X” is any conservative amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1.

[0185] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains at least one of the following substitutions: D25K, D25R, R32D, R32E, E96R, E96K, or E96E.

[0186] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains at least two of the following substitutions: D25K, D25R, R32D, R32E, E96R, E96K, or E96E.

[0187] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains at least three of the following substitutions: D25K, D25R, R32D, R32E, E96R, E96K, or E96E.

[0188] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 46U1197.70253WO00#1479354.297%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): D25K.

[0189] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): D25R.

[0190] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): R32D.

[0191] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): R32E.

[0192] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): E96R.

[0193] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): E96K.

[0194] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): E96E.47U1197.70253WO00#1479354.2

[0195] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): R32D and E96R.

[0196] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): R32D and E96E.

[0197] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): R32D and E96K.

[0198] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): R32E and E96R.

[0199] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): R32E and E96K.

[0200] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): D25K and E96R.

[0201] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino 48U1197.70253WO00#1479354.2acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): D25K and E96K.

[0202] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): D25R and E96R.

[0203] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following amino acid sequences of SEQ ID NOs: 2-15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-15, and which retains the following substitution(s): D25R and E96E.

[0204] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following sequences:

[0205] SEQ ID NO: 2 (designated herein as hIL-lOA), which contains an R32D substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSDVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0206] SEQ ID NOG (designated herein as hIL-lOB), which contains an E96R substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGRNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0207] SEQ ID NO:4 (designated herein as hIL-lOC), which contains an R32D and an E96R substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSDVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGRNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.49U1197.70253WO00#1479354.2

[0208] SEQ ID NO:5, which contains an R32E substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSEVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0209] SEQ ID NO:6, which contains an E96K substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGKNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0210] SEQ ID NO:7, which contains a D25R substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRRLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0211] SEQ ID NO:8, which contains a D25K substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0212] SEQ ID NO:9, which contains an R32D and E96K substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSDVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGKNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0213] SEQ ID NO: 10, which contains an R32E and E96R substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSEVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGRNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0214] SEQ ID NO: 11, which contains an R32E and E96K substitution relative to SEQ ID NO: 1:50U1197.70253WO00#1479354.2SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSEVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGKNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0215] SEQ ID NO: 12, which contains a D25K and E96R substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGRNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0216] SEQ ID NO: 13, which contains a D25K and E96K substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGKNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0217] SEQ ID NO: 14, which contains a D25R and E96R substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRRLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGRNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0218] SEQ ID NO: 15, which contains a D25R and E96E substitution relative to SEQ ID NO: 1:SPGQGTQSENSCTHFPGNLPNMLRRLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGKNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0219] In various other embodiments, the disclosure provides a variant IL- 10 cytokine that comprises any one of the following sequences:

[0220] SEQ ID NO: 16, which contains a D25X substitution relative to SEQ ID NO: 1, wherein “X” is any amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 16, and which retains the D25X substitution, wherein SEQ ID NO: 16 is:51U1197.70253WO00#1479354.2SPGQGTQSENSCTHFPGNLPNMLRXLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0221] SEQ ID NO: 17, which contains an R32X substitution relative to SEQ ID NO: 1, wherein “X” is any amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 17, and which retains the R32X substitution, wherein SEQ ID NO: 17 is:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSXVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0222] SEQ ID NO: 18, which contains an E96X substitution relative to SEQ ID NO: 1, wherein “X” is any amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 18, and which retains the E96X substitution, wherein SEQ ID NO: 18 is:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGXNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0223] SEQ ID NO: 19, which contains a D25X and E96X substitution relative to SEQ ID NO: 1, wherein “X” is any amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 19, and which retains the D25X and E96X substitutions, wherein SEQ ID NO: 19 is:SPGQGTQSENSCTHFPGNLPNMLRXLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGXNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0224] SEQ ID NO:20, which contains a D25X and R32X substitution relative to SEQ ID NO:1, wherein “X” is any amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least52U1197.70253WO00#1479354.290%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 20, and which retains the D25X and R32X substitutions, wherein SEQ ID NO: 20 is:SPGQGTQSENSCTHFPGNLPNMLRXLRDAFSXVKTFFQMKDQLDNLLLKESLLEDFKG YEGCQAESEMIQFYEEEVMPQAENQDPDIKAHVNSEGENEKTERERERRCHRFEPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0225] SEQ ID NO:21, which contains an R32X and an E96X substitution relative to SEQ ID NO: 1, wherein “X” is any amino acid substitution other than the wildtype amino acid at that position relative to SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 21, and which retains the R32X and E96X substitutions, wherein SEQ ID NO: 21 is:SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSXVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGXNLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.53U1197.70253WO00#1479354.2

Claims

CLAIMSWhat is claimed is:

1. A modified IL- 10 protein comprising at least one amino acid substitution at a position corresponding to D25, R32 and / or E96 of the wild-type IL- 10 protein of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and which retains the at least one amino acid substitution at a position corresponding to D25, R32 and / or E96 of SEQ ID NO: 1.

2. The modified IL- 10 protein according to claim 1, wherein the modified IL- 10 protein comprises a R32 substitution of the wild-type IL- 10 protein of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and which retains the R32D substitution.

3. The modified IL- 10 protein according to claim 1, wherein the modified IL- 10 protein comprises a E96 substitution of the wild-type IL- 10 protein of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and which retains the E96 substitution.

4. The modified IL- 10 protein according to claim 1, wherein the modified IL- 10 protein comprises a D25 substitution of the wild-type IL-10 protein of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and which retains the D25 substitution.

5. The modified IL- 10 protein according to claim 2, wherein the modified IL- 10 protein further comprises a E96 substitution of the wild-type IL- 10 protein of SEQ ID NO: 1.

6. The modified IL- 10 protein according to claim 4, wherein the modified IL- 10 protein further comprises a E96 substitution of the wild-type IL- 10 protein of SEQ ID NO: 1.54U1197.70253WO00#1479354.

27. The modified IL- 10 protein according to claim 1, wherein the modified IL- 10 protein comprises an amino acid substitution at amino acid R32, wherein the amino acid substitution is a D or E.

8. The modified IL- 10 protein according to claim 1, wherein the modified IL- 10 protein comprises an amino acid substitution at amino acid E96, wherein the amino acid substitution is a R or K.

9. The modified IL- 10 protein according to claim 1, wherein the modified IL- 10 protein comprises an amino acid substitution at amino acid D25, wherein the amino acid substitution is a R or K.

10. The modified IL- 10 protein according to claim 7, wherein the modified IL- 10 protein further comprises an amino acid substitution at amino acid E96, wherein the amino acid substitution is a R or K.

11. The modified IL- 10 protein according to claim 4 or claim 9, wherein the modified IL- 10 protein further comprises an amino acid substitution at amino acid E96, wherein the amino acid substitution is a R or K.

12. A modified IL- 10 protein comprising an amino acid substitution at a position corresponding to R32D and / or E96R of the wild-type IL- 10 protein of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and which retains the R32D and / or E96R substitutions.

13. A modified IL- 10 protein comprising an amino acid sequence selected from SEQ ID NOs: 2-21, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2-21, and which retains at least one substitution selected from the group consisting of: D25X, R32X, and E96X.55U1197.70253WO00#1479354.

214. An isolated nucleic acid encoding the modified IL-10 protein of any one of claims 1-13.

15. A vector comprising the isolated nucleic acid of claim 14.

16. A recombinant adeno associated virus (rAAV) comprising the vector of claim 15.

17. A recombinant adeno associated virus (rAAV) vector comprising a nucleic acid sequence encoding a modified IL- 10 protein, wherein the modified IL- 10 protein comprises an amino acid substitution at a position corresponding to D25, R32 and / or E96 of the wild-type IL- 10 protein of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and which retains at least one substitution selected from the group consisting of: D25, R32 and / or E96.

18. The rAAV of claim 16, wherein the modified IL- 10 protein comprises a R32D substitution, a E96R substitution, and / or a D25R substitution of the wild-type IL- 10 protein of SEQ ID NO: 1.

19. A pharmaceutical composition comprising the modified IL-10 protein of any one of claims 1-13 and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition comprising the rAAV of any one of claims 16-18 and a pharmaceutically acceptable carrier.

20. A method of producing the modified IL- 10 protein of any one of claims 1-13 by contacting a host cell with the rAAV of any one of claims 16-18.

21. The method of claim 20, wherein the host cell is a mammalian cell.

22. The method of claim 20, wherein the host cell is a HEK293 cell.

23. A method of treating an inflammatory disorder or an autoimmune disorder by administering to a subject an effective amount of the modified IL- 10 protein of any one of56U1197.70253WO00#1479354.2claims 1-13, the isolated nucleic acid of claim 14, the vector of claim 15, the rAAV of any one of claims 16-18, or the pharmaceutical composition of claims 19 or 20.

24. The method of claim 23, wherein the inflammatory or autoimmune disorder is colitis, sepsis, inflammatory bowel syndrome (IBD), rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), Grave’s disease, Sjorgren’s syndrome, Multiple Sclerosis, allergic asthma, asthma, or autoimmune lymphoproliferative syndrome.

25. The method of claim 23 or 24, wherein the subject is a mammal.

26. The method of claim 25, wherein the subject is human.

27. In a method of treating a patient by administering a modified IL- 10 protein, the improvement comprising the modified IL- 10 protein, wherein the modified IL- 10 protein retains normal anti-inflammatory effect, but has no or lower pro-inflammatory effects as compared to wild-type IL- 10 protein of SEQ ID NO: 1.

28. The method of claim 27, wherein the modified IL- 10 protein is of any one of claims 1- 13.

29. A method of treating a subject comprising: administering a modified IL- 10 protein; and(ii) that retains normal anti-inflammatory effect, but has no or lower pro-inflammatory effects as compared to wild-type IL- 10 protein of SEQ ID NO: 1.

30. The method of claim 29, wherein the modified IL-10 protein is of any one of claims 1- 13.

31. In a method of treating a patient by administering a rAAV vector comprising a nucleic acid sequence encoding a modified IL- 10 protein, the improvement comprising expressing the modified IL- 10 protein, wherein the modified IL- 10 protein retains normal anti-inflammatory effect, but has no or lower pro-inflammatory effects as compared to wild-type IL- 10 protein of SEQ ID NO: 1.57U1197.70253WO00#1479354.

232. The method of claim 31, wherein the rAAV is of any one of claims 16-18.

33. A method of treating a subject by administering a rAAV vector comprising: a transgene encoding a modified IL- 10 protein of any one of claims 1-13, wherein said modified IL- 10 protein retains normal anti-inflammatory effect, but has no or lower pro- inflammatory effects as compared to wild-type IL- 10 protein of SEQ ID NO: 1.

34. The method of claim 33, wherein the rAAV is of any one of claims 16-18.

35. The method of claim 33, wherein the modified IL- 10 protein is of any one of claims 1- 13.58U1197.70253WO00#1479354.2

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