CA3237037A1

CA3237037A1 - Multiple dosing with viral vectors

Info

Publication number: CA3237037A1
Application number: CA3237037A
Authority: CA
Inventors: Takashi Kei Kishimoto
Original assignee: Cartesian Therapeutics Inc
Current assignee: Cartesian Therapeutics Inc
Priority date: 2021-11-14
Filing date: 2022-11-14
Publication date: 2023-05-19
Also published as: JP2024545594A; WO2023086615A1; EP4429662A1

Abstract

This invention relates, at least in part, to repeated lower doses of a viral vector administered concomitantly with synthetic nanocarriers attached to an immunosuppressant, and related compositions.

Description

MULTIPLE DOSING WITH VIRAL VECTORS
RELATED APPLICATIONS
This application claims the benefit of priority under 35 U.S.C. 119(e) of U.S.
Provisional Application Serial Nos. 63/279,174, filed on November 14, 2021;
63/300,785, filed on January 19, 2022; 63/317,576, filed on March 8, 2022; and 63/338,672, filed on May 5, 2022; the entire contents of each of which are incorporated herein by reference.
SUMMARY OF THE INVENTION
This invention relates, at least in part, to repeated lower doses of a viral vector administered concomitantly with synthetic nanocarriers attached to an immunosuppressant, and related compositions. The compositions and methods provided herein can be used to result in efficacious transgene expression while being dose sparing so as to provide, for example, reduced toxicity and/or manufacturing benefits. In addition, it has been surprisingly found that multiple lower doses of a viral vector in a dosing can result in comparable transgene expression as a higher dose of the viral vector, such as a single higher dose, such as a dose of 1e1014 vector genomes/kg or 5e13 vector genomes/kg, with the administration (e.g., concomitant administration) of at least one dose of synthetic nanocarriers comprising an immunosuppressant in the dosing.
In one embodiment of any one of the compositions or methods provided herein, the repeated lower doses of a viral vector of a dosing are each lower than a dose of the viral vector that would be administered to a subject without the administration (e.g., concomitant administration) of synthetic nanocarriers comprising an immunosuppressant to achieve the same or comparable level of transgene expression in a comparable subject or test subject. In one embodiment of any one of the compositions or methods provided herein, the sum of the repeated lower doses of a viral vector of a dosing is less than half a dose of the viral vector administered to a subject without the administration (e.g., concomitant administration) of synthetic nanocarriers comprising an immunosuppressant to achieve the same or comparable level of transgene expression. In one embodiment of any one of the compositions or methods provided herein, each of the repeated lower doses of a viral vector of a dosing is at least 1/5 but less than a dose of the viral vector administered to a subject without the administration (e.g., concomitant administration) of synthetic nanocarriers comprising an immunosuppressant to achieve the same or comparable level of transgene expression. In one

- 2 -embodiment of any one of the compositions or methods provided herein, each of the repeated lower doses of a viral vector of a dosing is no more than 1/5 a dose of the viral vector administered to a subject without the administration (e.g., concomitant administration) of synthetic nanocarriers comprising an immunosuppressant to achieve the same or comparable level of transgene expression. In one embodiment of any one of the compositions or methods provided herein, each of the repeated lower doses of a viral vector of a dosing is 1/5 a dose of the viral vector administered to a subject without the administration (e.g., concomitant administration) of synthetic nanocarriers comprising an immunosuppressant to achieve the same or comparable level of transgene expression.
In one embodiment of any one of the compositions or methods provided herein, the lower dose of a viral vector administered to a subject is given otherwise under the same conditions as a dose of the viral vector administered without the synthetic nanocarriers comprising an immunosuppressant to achieve the same or comparable level of transgene expression to a comparable subject or test subject. In one embodiment of any one of the compositions or methods provided herein, the lower dose(s) of a viral vector of a dosing is concomitantly administered with synthetic nanocarriers comprising an immunosuppressant monthly or every other month.
In one embodiment of any one of the compositions or methods provided herein, the lower dose is lower than a dose of the viral vector administered without concomitant administration of the synthetic nanocarriers that are attached to an immunosuppressant but results in comparable transgene expression as the dose of the viral vector administered without concomitant administration of the synthetic nanocarriers that are attached to an immunosuppres sant.
In one embodiment of any one of the compositions or methods provided herein, a reduction in an undesired immune (eg., humoral) response to the viral vector and/or efficacious transgene or nucleic acid material expression and/or durable transgene or nucleic acid material expression and/or comparable transgene expression is the result of the dosing(s).
In an aspect, a method of manufacturing any one of the compositions or kits provided herein is provided. In one embodiment, the method of manufacturing comprises producing one or more doses or dosage forms of a viral vector and/or producing one or more doses or dosage forms of a population of synthetic nanocarriers that are attached to an immunosuppressant. In another embodiment of any one of the methods of manufacturing

- 3 -provided, the step of producing one or more doses or dosage forms of a population of synthetic nanocarriers that are attached to an immunosuppressant comprises attaching the immunosuppressant to synthetic nanocarriers. In another embodiment of any one of the methods of manufacturing provided, the method further comprises combining the one or more doses or dosage forms of the population of synthetic nanocarriers that are attached to an immunosuppressant and/or one or more doses or dosage forms of the viral vector in a kit.
In another aspect, a use of any one of the compositions or kits provided herein for the manufacture of a medicament for reducing or inhibiting an undesired immune response to a viral vector and/or provides durable and/or efficacious transgene or nucleic acid material expression in a subject is provided. In one embodiment, the composition or kit comprises one or more doses or dosage forms comprising a population of synthetic nanocarriers that are attached to an immunosuppressant and/or one or more doses or dosage forms comprising a viral vector, wherein the population of synthetic nanocarriers that are attached to an immunosuppressant and/or the viral vector are administered according to any one of the methods provided herein. In some embodiments of any one of the uses provided herein, the population of synthetic nanocarriers that are attached to an immunosuppressant comprises no viral vector antigen-presenting cell (APC) presentable antigens of the viral vector.
In another aspect, any one of the compositions or kits provided herein are provided for use in any one of the methods provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the expression with doses of viral vector and ImmTOR (PLA/PLA-PEG
synthetic nanocarriers comprising rapamycin).
DETAILED DESCRIPTION OF THE INVENTION
Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified materials or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting of the use of alternative terminology to describe the present invention.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

- 4 -As used in this specification and the appended claims, the singular forms "a,"
"an"
and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a polymer" includes a mixture of two or more such molecules or a mixture of differing molecular weights of a single polymer species, reference to "a synthetic nanocarrier" includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, reference to "a DNA molecule" includes a mixture of two or more such DNA molecules or a plurality of such DNA molecules, reference to "an immunosuppressant" includes a mixture of two or more such materials or a plurality of immunosuppressant molecules, and the like.
As used herein, the term "comprise" or variations thereof such as "comprises"
or "comprising" are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g.
features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprising" is inclusive and does not exclude additional, unrecited integers or method/process steps.
In embodiments of any one of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of' or "consisting of'. The phrase "consisting essentially of' is used herein to require the specified integer(s) or steps as well as .. those which do not materially affect the character or function of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) alone.
A. INTRODUCTION
Certain dosings provided herein can result in comparable, efficacious and/or durable transgene or nucleic acid material expression. The dosings in some embodiments may also result in dose sparing effects.
B. DEFINITIONS
"Administering" or "administration" or "administer" means providing a material to a subject in a manner that is pharmacologically useful. The term is intended to include

5 "causing to be administered" in some embodiments. "Causing to be administered"
means causing, urging, encouraging, aiding, inducing or directing, directly or indirectly, another party to administer the material.
"Administration schedule" refers to administration of dosings of one or more agents according to a determined schedule. The schedule can include the number of dosings as well as the frequency of such dosings or interval between dosings. Such an administration schedule may include a number of parameters that are varied to achieve a particular objective, preferably reduction of an undesired immune response to a viral vector antigen and/or efficacious and/or durable and/or comparable transgene or nucleic acid material expression. In embodiments, the administration schedule is any of the administration schedules as provided below in the Examples. In some embodiments, administration schedules according to the invention may be used to administer dosings to one or more test subjects. Immune responses and/or transgene or nucleic acid material expression in these test subjects can then be assessed to determine whether or not the schedule was effective in reducing an undesired immune response and/or efficacious and/or durable and/or comparable transgene or nucleic acid material expression. Whether or not a schedule had a desired effect can be determined using any of the methods provided herein or otherwise known in the art.
For example, a sample may be obtained from a subject to which dosings provided herein have been administered according to a specific administration schedule in order to determine whether or not specific immune cells, cytokines, antibodies, etc. were reduced, generated, activated, etc. and/or specific proteins or expression products were increased, reduced or generated, etc. Useful methods for detecting the presence and/or number of immune cells include, but are not limited to, flow cytometric methods (e.g., FACS), ELISpot, proliferation responses, cytokine production, and immunohistochemistry methods. Useful methods for determining the level of protein, such as antibody, production are well known in the art and include the assays provided herein. Such assays include ELISA assays.
Transgene expression levels can be assessed in a comparable subject or test subject.
One of ordinary skill in the art would understand that a "comparable subject"
is one in which an expression level can be determined and used as a comparator to a subject to be treated. A
"test subject" is any subject in which an expression level can be determined and from which the level can be a direct or indirect comparator, such as through scaling and/or extrapolation.
In some embodiments, a lower dose is determined by comparing a candidate lower dose in a comparable subjects or test subject with a higher dose administered to a comparable subject

- 6 -or test subject. The dose for a subject to be treated can then be determined through scaling or extrapolation.
In some embodiments, the lower dose is lower than a dose of the viral vector administered without concomitant administration of the synthetic nanocarriers that are attached to an immunosuppressant but that results in comparable transgene or nucleic acid material expression as the dose of the viral vector administered without concomitant administration of the synthetic nanocarriers that are attached to an immunosuppressant. The lower dose can be a single lower dose with the concomitant administration of synthetic nanocarriers comprising an immunosuppressant or more than one such lower doses over a finite period of time (e.g., 1 or 2 weeks) with at least one concomitant administration of synthetic nanocarriers that are attached to an immunosuppressant that results in comparable transgene or nucleic acid material expression. As used herein, "comparable transgene or nucleic acid material expression" refers to expression that is determined to not be statistically significantly different or that would not be expected to result in significant clinically different effects.
"Amount effective" in the context of a composition or dosage form for administration to a subject refers to an amount of the composition or dosage form that produces one or more desired immune responses or efficacious and/or durable and/or comparable transgene or nucleic acid material expression in the subject. Therefore, in some embodiments, an amount effective is any amount of a composition or dosage form, or combination thereof, provided herein that reduces an undesired immune response and/or provides efficacious and/or durable and/or comparable transgene or nucleic acid material expression. This amount can be for in vitro or in vivo purposes. For in vivo purposes, the amount can be one that a clinician would believe may have a clinical benefit for a subject as provided herein.
Amounts effective can involve reducing the level of an undesired immune response, although in some embodiments, it involves preventing an undesired immune response altogether. Amounts effective can also involve delaying the occurrence of an undesired immune response. An amount that is effective can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic result. Amounts effective, preferably, result in a reduction in an undesired immune response in a subject specific to a viral vector and/or provides efficacious and/or durable and/or comparable transgene or nucleic acid material expression of a viral vector. Amounts effective, can also result in a tolerogenic immune response in a subject to an antigen, such as a viral vector antigen. In other embodiments, the

- 7 -amounts effective can involve enhancing the level of a desired response, such as a therapeutic endpoint or result. The achievement of any of the foregoing can be monitored by routine methods.
In some embodiments of any one of the compositions and methods provided, the amount effective is one in which the desired immune response persists in the subject for at least 1 week, at least 2 weeks, at least 1 month, or longer. In other embodiments of any of the compositions and methods provided, the amount effective is one which produces a measurable desired response, for example, a measurable desired immune response, such as a decrease in a humoral immune response (e.g., to a specific antigen) and/or transgene or nucleic acid material expression response, for at least 1 week, at least 2 weeks, at least 1 month, or longer.
Amounts effective will depend, of course, on the particular subject being treated; the severity of a condition, disease or disorder; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reason.
Doses of the synthetic nanocarriers attached to an immunosuppressant can refer to the amount of the immunosuppressant attached to the synthetic nanocarriers.
Alternatively, the dose can be administered based on the number of synthetic nanocarriers that provide the desired amount of immunosuppressants.
"Anti-viral vector immune response" or "immune response against a viral vector" or the like refers to any undesired immune response against a viral vector. In some embodiments, the undesired immune response is an antigen-specific immune response against the viral vector or an antigen thereof. In some embodiments, the immune response is specific to a viral antigen of the viral vector. The immune response may be an anti-viral vector antibody response, an anti-viral vector T cell immune response, such as a CD4+ T cell or CD8+ T cell immune response, or an anti-viral vector B cell immune response.

- 8 -"Antigen" means a B cell antigen or T cell antigen. "Type(s) of antigens"
means molecules that share the same, or substantially the same, antigenic characteristics. In some embodiments, antigens may be proteins, polypeptides, peptides, lipoproteins, glycolipids, polynucleotides, polysaccharides or are contained or expressed in cells. In some embodiments, such as when the antigens are not well defined or characterized, the antigens may be contained within a cell or tissue preparation, cell debris, cell exosomes, conditioned media, etc.
"Antigen-specific" refers to any immune response that results from the presence of the antigen, or portion thereof, or that generates molecules that specifically recognize or bind the antigen. In some embodiments, when the antigen is of a viral vector, antigen-specific may mean viral vector-specific. For example, where the immune response is antigen-specific antibody production, such as viral vector-specific antibody production, antibodies are produced that specifically bind the antigen (e.g., viral vector). As another example, where the immune response is antigen-specific B cell or CD4+ T cell proliferation and/or activity, the proliferation and/or activity results from recognition of the antigen, or portion thereof, alone or in complex with MHC molecules, B cells, etc.
"Assessing an immune response" refers to any measurement or determination of the level, presence or absence, reduction, increase in, etc. of an immune response in vitro or in vivo. Such measurements or determinations may be performed on one or more samples obtained from a subject. Such assessing can be performed with any of the methods provided herein or otherwise known in the art.
"Attach" or "Attached" or "Couple" or "Coupled" (and the like) means to chemically associate one entity (for example a moiety) with another. In some embodiments, the attaching is covalent, meaning that the attachment occurs in the context of the presence of a covalent bond between the two entities. In non-covalent embodiments, the non-covalent attaching is mediated by non-covalent interactions including but not limited to charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. In embodiments, encapsulation is a form of attaching. In embodiments, the viral vector and synthetic nanocarriers attached to an immunosuppressant are not attached to one another, meaning that the viral vector and

- 9 -synthetic nanocarriers attached to an immunosuppressant are not subjected to a process specifically intended to chemically associate one with another.
An "at risk" subject is one in which a health practitioner believes has a chance of having a disease, disorder or condition or is one a health practitioner believes has a chance of experiencing an undesired immune response as provided herein and would benefit from the compositions and methods provided. In some embodiments, the subjects are those that would benefit from administration of a viral vector.
"Average", as used herein, refers to the arithmetic mean unless otherwise noted.
"Concomitantly" means administering two or more materials/agents to a subject in a manner that is correlated in time, preferably sufficiently correlated in time so as to provide a modulation in an immune or physiologic response, and even more preferably the two or more materials/agents are administered in combination. In embodiments, concomitant administration may encompass administration of two or more materials/agents within a specified period of time, preferably within 1 month, more preferably within 1 week, still more preferably within 1 day, and even more preferably within 1 hour. In embodiments, the materials/agents may be repeatedly administered concomitantly, that is concomitant administration on more than one occasion, such as as may be provided in the Examples.
"Determining" or "determine" means to ascertain a factual relationship.
Determining may be accomplished in a number of ways, including but not limited to performing experiments, or making projections. For instance, a dose of an immunosuppressant and/or viral vector, may be determined by starting with a test dose and using known scaling techniques (such as allometric or isometric scaling) to determine the dose for administration.
Such may also be used to determine a protocol or administration schedule as provided herein.
In another embodiment, the dose may be determined by testing various doses in a subject, i.e., through direct experimentation based on experience and guiding data. In embodiments, "determining" or "determine" comprises "causing to be determined." "Causing to be determined" means causing, urging, encouraging, aiding, inducing or directing or acting in coordination with an entity for the entity to ascertain a factual relationship; including directly or indirectly, or expressly or impliedly.
"Dose" refers to a specific quantity of a pharmacologically and/or immunologically active material for administration to a subject for a given time. In general, doses of the synthetic nanocarriers comprising an immunosuppressant and/or viral vectors in the methods and compositions of the invention refer to the amount of the synthetic nanocarriers

- 10 -comprising an immunosuppressant and/or viral vectors. Alternatively, the dose can be administered based on the number of synthetic nanocarriers that provide the desired amount of an immunosuppressant, in instances when referring to a dose of synthetic nanocarriers that comprise an immunosuppressant. When dose is used in the context of a repeated dosing, dose refers to the amount of each of the repeated doses, which may be the same or different.
"Dosing" means the administration of a pharmacologically and/or immunologically active material or combination of pharmacologically and/or immunologically active materials to a subject. The materials of a dosing may be administered concomitantly in any one of the methods provided herein. The materials of a dosing may be administered separately in separate compositions in any one of the methods provided herein.
"Encapsulate" means to enclose at least a portion of a substance within a synthetic nanocarrier. In some embodiments, a substance is enclosed completely within a synthetic nanocarrier. In other embodiments, most or all of a substance that is encapsulated is not exposed to the local environment external to the synthetic nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed to the local environment. Encapsulation is distinct from absorption, which places most or all of a substance on a surface of a synthetic nanocarrier, and leaves the substance exposed to the local environment external to the synthetic nanocarrier. In any one of the methods or composition provided herein, the immunosuppressant may be encapsulated in the synthetic nanocarriers.
"Expression control sequences" are any sequences that can affect expression and can include promoters, enhancers, and operators. In one embodiment of any one of the methods or compositions provided, the expression control sequence is a promoter. In one embodiment of any one of the methods or compositions provided, the expression control sequence is a liver-specific promoter or a constitutive promoter. "Liver-specific promoters"
are those that exclusively or preferentially result in expression in cells of the liver.
"Constitutive promoters" are those that are thought of being generally active and not exclusive or preferential to certain cells. In any one of the nucleic acids or viral vectors provided herein the promoter may be any one of the promoters provided herein.
"Generating" means causing an action, such as an immune or physiologic response (e.g., a tolerogenic immune response) to occur, either directly oneself or indirectly.
"Identifying a subject" is any action or set of actions that allows a clinician to recognize a subject as one who may benefit from the methods, compositions or kits provided

- 11 -herein. Preferably, the identified subject is one who is in need of a therapeutic benefit from a viral vector and in which an undesired immune response is expected to occur as provided herein. The action or set of actions may be either directly oneself or indirectly. In one embodiment of any one of the methods provided herein, the method further comprises identifying a subject in need of a method, composition or kit as provided herein.
"Immunosuppressant" means a compound that causes an APC to have an immunosuppressive effect (e.g., tolerogenic effect) or a T cell or a B cell to be suppressed.
An immunosuppressive effect generally refers to the production or expression of cytokines or other factors by the APC that reduces, inhibits or prevents an undesired immune response or that promotes a desired immune response, such as a regulatory immune response.
When the APC acquires an immunosuppressive function (under the immunosuppressive effect) on immune cells that recognize an antigen presented by this APC, the immunosuppressive effect is said to be specific to the presented antigen. Without being bound by any particular theory, it is thought that the immunosuppressive effect is a result of the immunosuppressant being delivered to the APC, preferably in the presence of an antigen. In one embodiment, the immunosuppressant is one that causes an APC to promote a regulatory phenotype in one or more immune effector cells. For example, the regulatory phenotype may be characterized by the inhibition of the production, induction, stimulation or recruitment of antigen-specific CD4+ T cells or B cells, the inhibition of the production of antigen-specific antibodies, the production, induction, stimulation or recruitment of Treg cells (e.g., CD4+CD25highFoxP3+
Treg cells), etc. This may be the result of the conversion of CD4+ T cells or B cells to a regulatory phenotype. This may also be the result of induction of FoxP3 in other immune cells, such as CD8+ T cells, macrophages and iNKT cells. In one embodiment, the immunosuppressant is one that affects the response of the APC after it processes an antigen.
In another embodiment, the immunosuppressant is not one that interferes with the processing of the antigen. In a further embodiment, the immunosuppressant is not an apoptotic-signaling molecule. In another embodiment, the immunosuppressant is not a phospholipid.
Immunosuppressants include, but are not limited to, statins; mTOR inhibitors, such as rapamycin or a rapamycin analog; TGF-P signaling agents; TGF-P receptor agonists; histone deacetylase inhibitors, such as Trichostatin A; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-i3 inhibitors, such as 6Bio, Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2), such as Misoprostol; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor (PDE4),

- 12 -such as Rolipram; proteasome inhibitors; kinase inhibitors; G-protein coupled receptor agonists; G-protein coupled receptor antagonists; glucocorticoids; retinoids;
cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators;
peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3KB
inhibitors, such as TGX-221; autophagy inhibitors, such as 3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidized ATPs, such as P2X
receptor blockers.
Immunosuppressants also include IDO, vitamin D3, cyclosporins, such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol, methotrexate and triptolide.
In embodiments, the immunosuppressant may comprise any of the agents provided herein.
The immunosuppressant can be a compound that directly provides the immunosuppressive effect on APCs or it can be a compound that provides the immunosuppressive effect indirectly (i.e., after being processed in some way after administration). Immunosuppressants, therefore, include prodrug forms of any of the compounds provided herein.
In embodiments of any one of the methods, compositions or kits provided herein, the immunosuppressants provided herein are attached to synthetic nanocarriers. In preferable embodiments, the immunosuppressant is an element that is in addition to the material that makes up the structure of the synthetic nanocarrier. For example, in one embodiment, where the synthetic nanocarrier is made up of one or more polymers, the immunosuppressant is a compound that is in addition and attached to the one or more polymers. As another example, in one embodiment, where the synthetic nanocarrier is made up of one or more lipids, the immunosuppressant is again in addition and attached to the one or more lipids.
In embodiments, such as where the material of the synthetic nanocarrier also results in an immunosuppressive effect, the immunosuppressant is an element present in addition to the material of the synthetic nanocarrier that results in an immunosuppressive effect.
Other exemplary immunosuppressants include, but are not limited, small molecule drugs, natural products, antibodies (e.g., antibodies against CD20, CD3, CD4), biologics-based drugs, carbohydrate-based drugs, nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab;
anti-CD3;
tacrolimus (FK506); cytokines and growth factors, such as TGF-f3 and IL-10;
etc. Further

- 13 -immunosuppressants, are known to those of skill in the art, and the invention is not limited in this respect.
"Load", when attached to a synthetic nanocarrier, is the amount of the immunosuppressant attached to a synthetic nanocarrier based on the total dry recipe weight of materials in an entire synthetic nanocarrier (weight/weight). Generally, such a load is calculated as an average across a population of synthetic nanocarriers. In one embodiment, the load of the immunosuppressant on average across the synthetic nanocarriers is between 0.1% and 99%. In another embodiment, the load is between 0.1% and 50%. In yet another embodiment, the load of the immunosuppressant is between 0.1% and 20%. In a further embodiment, the load of the immunosuppressant is between 0.1% and 10%. In still a further embodiment, the load of the immunosuppressant is between 1% and 10%. In still a further embodiment, the load of the immunosuppressant is between 7% and 20%. In yet another embodiment, the load of the immunosuppressant is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19% or at least 20%, at least 25%, or at least 30% on average across the population of synthetic nanocarriers. In yet a further embodiment, the load of the immunosuppressant is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% on average across the population of synthetic nanocarriers. In some embodiments of the above embodiments, the load of the immunosuppressant is no more than 25% or 30% on average across a population of synthetic nanocarriers. In embodiments, the load is calculated as may be described in the Examples or as otherwise known in the art.
"Maximum dimension of a synthetic nanocarrier" means the largest dimension of a nanocarrier measured along any axis of the synthetic nanocarrier. "Minimum dimension of a synthetic nanocarrier" means the smallest dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spheroidal synthetic nanocarrier, the maximum and minimum dimension of a synthetic nanocarrier would be substantially identical, and would be the size of its diameter. Similarly, for a cuboidal synthetic nanocarrier, the minimum dimension of a synthetic nanocarrier would be the smallest of its height, width or length, while the maximum dimension of a synthetic

- 14 -nanocarrier would be the largest of its height, width or length. In an embodiment, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm. In an embodiment, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 5 rim. Preferably, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is greater than 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm, and more preferably still greater than 150 nm. Aspects ratios of the maximum and minimum dimensions of synthetic nanocarriers may vary depending on the embodiment. For instance, aspect ratios of the maximum to minimum dimensions of the synthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferably from 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and yet more preferably from 1:1 to 10:1. Preferably, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or less than 3 m, more preferably equal to or less than 2 m, more preferably equal to or less than 1 m, more preferably equal to or less than 800 nm, more preferably equal to or less than 600 nm, and more preferably still equal to or less than 500 nm. In preferred embodiments, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, more preferably equal to or greater than 140 nm, and more preferably still equal to or greater than 150 nm. Measurement of synthetic nanocarrier dimensions (e.g., effective diameter) may be obtained, in some embodiments, by suspending the synthetic nanocarriers in a liquid (usually aqueous) media and using dynamic light scattering (DLS) (e.g., using a Brookhaven ZetaPALS instrument). For example, a suspension of synthetic nanocarriers can be diluted from an aqueous buffer into purified water to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01 to 0.1 mg/mL. The diluted suspension may be prepared directly inside, or transferred to, a suitable cuvette for DLS analysis. The cuvette may then be placed in the DLS, allowed to

15 equilibrate to the controlled temperature, and then scanned for sufficient time to acquire a stable and reproducible distribution based on appropriate inputs for viscosity of the medium and refractive indicies of the sample. The effective diameter, or mean of the distribution, is then reported. Determining the effective sizes of high aspect ratio, or non-spheroidal, synthetic nanocarriers may require augmentative techniques, such as electron microscopy, to obtain more accurate measurements. "Dimension" or "size" or "diameter" of synthetic nanocarriers means the mean of a particle size distribution, for example, obtained using dynamic light scattering.
"Non-methoxy-terminated polymer" means a polymer that has at least one terminus that ends with a moiety other than methoxy. In some embodiments, the polymer has at least two termini that ends with a moiety other than methoxy. In other embodiments, the polymer has no termini that ends with methoxy. "Non-methoxy-terminated, pluronic polymer" means a polymer other than a linear pluronic polymer with methoxy at both termini.
Polymeric nanoparticles as provided herein can comprise non-methoxy-terminated polymers or non-methoxy-terminated, pluronic polymers.
"Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier"
means a pharmacologically inactive material used together with a pharmacologically active material to formulate the compositions. Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers.
"Providing" means an action or set of actions that an individual performs that supply a needed item or set of items or methods for practicing of the present invention. The action or set of actions may be taken either directly oneself or indirectly.
"Providing a subject" is any action or set of actions that causes a clinician to come in contact with a subject and administer a composition provided herein thereto or to perform a method provided herein thereupon. In some embodiments, the subject is one who is in need of viral vector administration and antigen-specific immune tolerance thereto or any one of the desired results as provided herein. The action or set of actions may be taken either directly oneself or indirectly. In one embodiment of any one of the methods provided herein, the method further comprises providing a subject.
"Subject" means animals, including warm blooded mammals such as humans and primates; avians; domestic household or farm animals such as cats, dogs, sheep, goats, cattle,

- 16 -horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish;
reptiles; zoo and wild animals; and the like. As used herein, a subject may be in one need of any one of the methods or compositions provided herein.
"Synthetic nanocarrier(s)" means a discrete object that is not found in nature, and that possesses at least one dimension that is less than or equal to 5 microns in size. Albumin nanoparticles are generally included as synthetic nanocarriers, however in certain embodiments the synthetic nanocarriers do not comprise albumin nanoparticles.
In embodiments, synthetic nanocarriers do not comprise chitosan. In other embodiments, synthetic nanocarriers are not lipid-based nanoparticles. In further embodiments, synthetic nanocarriers do not comprise a phospholipid.
A synthetic nanocarrier can be, but is not limited to, one or a plurality of lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles where the majority of the material that makes up their structure are lipids), polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles that are primarily made up of viral structural proteins but that are not infectious or have low infectivity), peptide or protein-based particles (also referred to herein as protein particles, i.e., particles where the majority of the material that makes up their structure are peptides or proteins) (such as albumin nanoparticles) and/or nanoparticles that are developed using a combination of nanomaterials such as lipid-polymer nanoparticles.
.. Synthetic nanocarriers may be a variety of different shapes, including but not limited to spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like.
Synthetic nanocarriers according to the invention comprise one or more surfaces.
Exemplary synthetic nanocarriers that can be adapted for use in the practice of the present invention comprise: (1) the biodegradable nanoparticles disclosed in US Patent 5,543,158 to Gref et al., (2) the polymeric nanoparticles of Published US Patent Application 20060002852 to Saltzman et al., (3) the lithographically constructed nanoparticles of Published US Patent Application 20090028910 to DeSimone et al., (4) the disclosure of WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosed in Published US Patent Application 2008/0145441 to Penades et al., (6) the protein nanoparticles disclosed in Published US Patent Application 20090226525 to de los Rios et al., (7) the virus-like particles disclosed in published US
Patent Application 20060222652 to Sebbel et al., (8) the nucleic acid attached virus-like particles disclosed in published US Patent Application 20060251677 to Bachmann et al., (9) the virus-like particles disclosed in W02010047839A1 or W02009106999A2, (10) the

- 17 -nanoprecipitated nanoparticles disclosed in P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles"
Nanomedicine. 5(6):843-853 (2010), (11) apoptotic cells, apoptotic bodies or the synthetic or semisynthetic mimics disclosed in U.S. Publication 2002/0086049, or (12) those of Look et al., Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice" J. Clinical Investigation 123(4):1741-1749(2013). In embodiments, synthetic nanocarriers may possess an aspect ratio greater than or equal to 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.
Synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface with hydroxyl groups that activate complement or alternatively comprise a surface that consists essentially of moieties that are not hydroxyl groups that activate complement. In a preferred embodiment, synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that substantially activates complement or alternatively comprise a surface that consists essentially of moieties that do not substantially activate complement. In a more preferred embodiment, synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that activates complement or alternatively comprise a surface that consists essentially of moieties that do not activate complement. In embodiments, synthetic nanocarriers exclude virus-like particles. In embodiments, synthetic nanocarriers may possess an aspect ratio greater than or equal to 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.
"Transgene or nucleic acid material expression" refers to the level of the transgene or nucleic acid material expression product of a viral vector in a subject, the transgene or nucleic acid material being delivered by the viral vector. In some embodiments, the level of expression may be determined by measuring transgene protein concentrations in various tissues or systems of interest in the subject, a comparable subject or a test subject or as between two sets of comparable subjects or test subjects. Alternatively, when the expression product is a nucleic acid, the level of expression may be measured by nucleic acid products.
Increasing expression can be determined, for example, by measuring the amount of the expression product in a sample obtained from a subject and comparing it to a prior sample.
Durability of expression may be measured by similar or other methods that would be

- 18 -apparent to one of ordinary skill in the art. The sample may be a tissue sample. In some embodiments, the expression product can be measured using flow cytometry.
"Undesired immune response" refers to any undesired immune response that results from exposure to an antigen, promotes or exacerbates a disease, disorder or condition provided herein (or a symptom thereof), or is symptomatic of a disease, disorder or condition provided herein. Such immune responses generally can have a negative impact on a subject's health or can be symptomatic of a negative impact on a subject's health.
Undesired immune responses may be undesired humoral immune responses, which can include antigen-specific antibody production, antigen-specific B cell proliferation and/or activity or antigen-specific CD4+ T cell proliferation and/or activity. Generally, herein, these undesired immune responses are specific to a viral vector and counteract the beneficial effects desired of administration with the viral vector.
"Viral vector" means a vector construct with viral components, such as capsid and/or coat proteins, that has been adapted to comprise and deliver a transgene or nucleic acid material that encodes a therapeutic, such as a therapeutic protein or nucleic acid, which transgene or nucleic acid material can be expressed as provided herein.
"Expressed" or "expression" or the like refers to the synthesis of a functional (i.e., physiologically active for the desired purpose) product after the transgene or nucleic acid material is transduced into a cell and processed by the transduced cell. Such a product is also referred to herein as an "expression product". Viral vectors can be based on, without limitation, adeno-associated viruses, such as AAV8 or AAV2. Thus, an AAV vector provided herein is a viral vector based on an AAV, such as AAV8 or AAV2, and has viral components, such as a capsid and/or coat protein, therefrom that can package for delivery the transgene or nucleic acid material. In some embodiments, the viral vector is a "chimeric viral vector".
In such embodiments, this means that the viral vector is made up of viral components that are derived from more than one virus or viral vector.
"Viral vector antigen" means an antigen that is associated with a viral vector (i.e., the viral vector or a fragment thereof that can generate an immune response against the viral vector (e.g., the production of anti-viral vector-specific antibodies)). Viral vector antigens may be presented for recognition by the immune system (e.g., cells of the immune system, such as presented by antigen presenting cells, including but not limited to dendritic cells, B
cells or macrophages). The viral vector antigens may be presented for recognition by, for example, T cells. Such antigens may be recognized by and trigger an immune response in a

- 19 -T cell via presentation of an epitope of the antigen bound to a Class I or Class II major histocompatability complex molecule (MHC). Viral vector antigens generally include proteins, polypeptides, peptides, polynucleotides, etc., or are contained or expressed in, on or by cells. The viral vector antigens, in some embodiments, comprise MHC Class I-restricted epitopes and/or MHC Class II-restricted epitopes and/or B cell epitopes. In some embodiments, one or more tolerogenic immune responses specific to the viral vector results with the methods, compositions or kits provided herein. In embodiments, populations of the synthetic nanocarriers comprise no added viral vector antigens, meaning that no substantial amounts of viral vector antigens are intentionally added to the synthetic nanocarriers during the manufacturing thereof.
C. COMPOSITIONS USEFUL IN THE PRACTICE OF THE METHODS
The transgene or nucleic acid material, such as of the viral vectors, provided herein may encode any protein or portion thereof or nucleic acid or portion thereof beneficial to a subject, such as one with a disease or disorder. In embodiments, the subject has or is suspected of having a disease or disorder whereby the subject's endogenous version of the protein is defective or produced in limited amounts or not at all. The subject may be one with any one of the diseases or disorders as provided herein, and the transgene or nucleic acid material is one that encodes any one of the therapeutic proteins or portion thereof as provided herein. The transgene or nucleic acid material provided herein may encode a functional version of any protein that through some defect in the endogenous version of which in a subject (including a defect in the expression of the endogenous version) results in a disease or disorder in the subject.
The sequence of a transgene or nucleic acid material may also include an expression .. control sequence. Expression control sequences include promoters, enhancers, and operators, and are generally selected based on the expression systems in which the expression construct is to be utilized. In some embodiments, promoter and enhancer sequences are selected for the ability to increase gene expression, while operator sequences may be selected for the ability to regulate gene expression. The transgene may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. The transgene may also include sequences that are necessary for replication in a host cell.
Exemplary expression control sequences include liver-specific promoter sequences and constitutive promoter sequences, such as any one that may be provided herein.

- 20 -Generally, promoters are operatively linked upstream (i.e., 5') of the sequence coding for a desired expression product. The transgene also may include a suitable polyadenylation sequence operably linked downstream (i.e., 3') of the coding sequence.
Viruses have evolved specialized mechanisms to transport their genomes inside the cells that they infect; viral vectors based on such viruses can be tailored to transduce cells to specific applications. Examples of viral vectors that may be used as provided herein are known in the art or described herein. Suitable viral vectors include, for instance, adeno-associated virus (AAV)-based vectors.
The viral vectors provided herein can be based on adeno-associated viruses (AAVs).
AAV vectors have been of particular interest for use in therapeutic applications such as those described herein. AAV is a DNA virus, which is not known to cause human disease.
Generally, AAV requires co-infection with a helper virus (e.g., an adenovirus or a herpes virus), or expression of helper genes, for efficient replication. For a description of AAV-based vectors, see, for example, U.S. Pat. Nos. 8,679,837, 8,637,255, 8,409,842, 7,803,622, and 7,790,449, and U.S. Publication Nos. 20150065562, 20140155469, 20140037585, 20130096182, 20120100606, and 20070036757. The AAV vectors may be recombinant AAV vectors. The AAV vectors may also be self-complementary (sc) AAV vectors, which are described, for example, in U.S. Patent Publications 2007/01110724 and 2004/0029106, and U.S. Pat. Nos. 7,465,583 and 7,186,699.
The adeno-associated virus on which a viral vector is based may be of a specific serotype, such as AAV8 or AAV2. In some embodiments of any one of the methods or compositions provided herein, therefore, the AAV vector is an AAV8 or AAV2 vector.
A wide variety of synthetic nanocarriers can be used to attach to immunosuppressants of the dosings. In some embodiments, synthetic nanocarriers are spheres or spheroids. In some embodiments, synthetic nanocarriers are flat or plate-shaped. In some embodiments, synthetic nanocarriers are cubes or cubic. In some embodiments, synthetic nanocarriers are ovals or ellipses. In some embodiments, synthetic nanocarriers are cylinders, cones, or pyramids.
In some embodiments, it is desirable to use a population of synthetic nanocarriers that is relatively uniform in terms of size or shape so that each synthetic nanocarrier has similar properties. For example, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers, based on the total number of synthetic nanocarriers, may have a minimum

- 21 -dimension or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension of the synthetic nanocarriers.
Synthetic nanocarriers can be solid or hollow and can comprise one or more layers. In some embodiments, each layer has a unique composition and unique properties relative to the other layer(s). To give but one example, synthetic nanocarriers may have a core/shell structure, wherein the core is one layer (e.g., a polymeric core) and the shell is a second layer (e.g., a lipid bilayer or monolayer). Synthetic nanocarriers may comprise a plurality of different layers.
In some embodiments, synthetic nanocarriers may optionally comprise one or more lipids. In some embodiments, a synthetic nanocarrier may comprise a liposome.
In some embodiments, a synthetic nanocarrier may comprise a lipid bilayer. In some embodiments, a synthetic nanocarrier may comprise a lipid monolayer. In some embodiments, a synthetic nanocarrier may comprise a micelle. In some embodiments, a synthetic nanocarrier may comprise a core comprising a polymeric matrix surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some embodiments, a synthetic nanocarrier may comprise a non-polymeric core (e.g., metal particle, quantum dot, ceramic particle, bone particle, viral particle, proteins, nucleic acids, carbohydrates, etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).
In other embodiments, synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc. In some embodiments, a non-polymeric synthetic nanocarrier is an aggregate of non-polymeric components, such as an aggregate of metal atoms (e.g., gold atoms).
In some embodiments, synthetic nanocarriers may optionally comprise one or more amphiphilic entities. In some embodiments, an amphiphilic entity can promote the production of synthetic nanocarriers with increased stability, improved uniformity, or increased viscosity. In some embodiments, amphiphilic entities can be associated with the interior surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers in accordance with the present invention. Such amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine (DPPC);
dioleylphosphatidyl ethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol;
diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such

- 22 -as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides;
fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span 85) glycocholate;
sorbitan monolaurate (Span 20); polysorbate 20 (Tween 20); polysorbate 60 (Tween 60);
polysorbate 65 (Tween 65); polysorbate 80 (Tween 80); polysorbate 85 (Tween 85);
polyoxyethylene monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine;
phosphatidylinositol;sphingomyelin; phosphatidylethanolamine (cephalin);
cardiolipin;
phosphatidic acid; cerebrosides; dicetylphosphate;
dipalmitoylphosphatidylglycerol;
stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerol ricinoleate;
hexadecyl sterate; isopropyl myristate; tyloxapol; poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethylene glycol)400-monostearate;
phospholipids; synthetic and/or natural detergents having high surfactant properties; deoxycholates;
cyclodextrins;
chaotropic salts; ion pairing agents; and combinations thereof. An amphiphilic entity component may be a mixture of different amphiphilic entities. Those skilled in the art will recognize that this is an exemplary, not comprehensive, list of substances with surfactant activity. Any amphiphilic entity may be used in the production of synthetic nanocarriers to be used in accordance with the present invention.
In some embodiments, synthetic nanocarriers may optionally comprise one or more carbohydrates. Carbohydrates may be natural or synthetic. A carbohydrate may be a derivatized natural carbohydrate. In certain embodiments, a carbohydrate comprises monosaccharide or disaccharide, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethylstarch, carageenan, glycon, amylose, chitosan, N,0-carboxylmethylchitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In embodiments, the synthetic nanocarriers do not comprise (or specifically exclude) carbohydrates, such as a polysaccharide. In certain embodiments, the carbohydrate may comprise a carbohydrate derivative such as a sugar

-23 -alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.
In some embodiments, synthetic nanocarriers can comprise one or more polymers.
In some embodiments, the synthetic nanocarriers comprise one or more polymers that is a non-methoxy-terminated, pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated, pluronic polymers. In some embodiments, all of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated, pluronic polymers. In some embodiments, the synthetic nanocarriers comprise one or more polymers that is a non-methoxy-terminated polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated polymers. In some embodiments, all of the polymers that make up the synthetic nanocarriers are non-methoxy-terminated polymers. In some embodiments, the synthetic nanocarriers comprise one or more polymers that do not comprise pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up the synthetic nanocarriers do not comprise pluronic polymer. In some embodiments, all of the polymers that make up the synthetic nanocarriers do not comprise pluronic polymer. In some embodiments, such a polymer can be surrounded by a coating layer (e.g., liposome, lipid monolayer, micelle, etc.).
In some embodiments, various elements of the synthetic nanocarriers can be attached to the polymer.
The immunosuppressants can be attached to the synthetic nanocarriers by any of a number of methods. Generally, the attaching can be a result of bonding between the immunosuppressants and the synthetic nanocarriers. This bonding can result in the immunosuppressants being attached to the surface of the synthetic nanocarriers and/or contained (encapsulated) within the synthetic nanocarriers. In some embodiments, however, the immunosuppressants are encapsulated by the synthetic nanocarriers as a result of the structure of the synthetic nanocarriers rather than bonding to the synthetic nanocarriers. In preferable embodiments, the synthetic nanocarrier comprises a polymer as provided herein, and the immunosuppressants are attached to the polymer.

- 24 -When attaching occurs as a result of bonding between the immunosuppressants and synthetic nanocarriers, the attaching may occur via a coupling moiety. A
coupling moiety can be any moiety through which an immunosuppressant is bonded to a synthetic nanocarrier.
Such moieties include covalent bonds, such as an amide bond or ester bond, as well as separate molecules that bond (covalently or non-covalently) the immunosuppressant to the synthetic nanocarrier. Such molecules include linkers or polymers or a unit thereof. For example, the coupling moiety can comprise a charged polymer to which an immunosuppressant electrostatically binds. As another example, the coupling moiety can comprise a polymer or unit thereof to which it is covalently bonded.
In preferred embodiments, the synthetic nanocarriers comprise a polymer as provided herein. These synthetic nanocarriers can be completely polymeric or they can be a mix of polymers and other materials.
In some embodiments, the polymers of a synthetic nanocarrier associate to form a polymeric matrix. In some of these embodiments, a component, such as an immunosuppressant, can be covalently associated with one or more polymers of the polymeric matrix. In some embodiments, covalent association is mediated by a linker. In some embodiments, a component can be noncovalently associated with one or more polymers of the polymeric matrix. For example, in some embodiments, a component can be encapsulated within, surrounded by, and/or dispersed throughout a polymeric matrix.
Alternatively or additionally, a component can be associated with one or more polymers of a polymeric matrix by hydrophobic interactions, charge interactions, van der Waals forces, etc.
A wide variety of polymers and methods for forming polymeric matrices therefrom are known conventionally.
Polymers may be natural or unnatural (synthetic) polymers. Polymers may be .. homopolymers or copolymers comprising two or more monomers. In terms of sequence, copolymers may be random, block, or comprise a combination of random and block sequences. Typically, polymers in accordance with the present invention are organic polymers.
In some embodiments, the polymer comprises a polyester, polycarbonate, polyamide, or polyether, or unit thereof. In other embodiments, the polymer comprises poly(ethylene glycol) (PEG), polypropylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or a polycaprolactone, or unit thereof. In some embodiments, it is preferred that the polymer is biodegradable. Therefore, in these embodiments, it is preferred that if the

- 25 -polymer comprises a polyether, such as poly(ethylene glycol) or polypropylene glycol or unit thereof, the polymer comprises a block-co-polymer of a polyether and a biodegradable polymer such that the polymer is biodegradable. In other embodiments, the polymer does not solely comprise a polyether or unit thereof, such as poly(ethylene glycol) or polypropylene glycol or unit thereof.
Other examples of polymers suitable for use in the present invention include, but are not limited to polyethylenes, polycarbonates (e.g. poly(1,3-dioxan-20ne)), polyanhydrides (e.g. poly(sebacic anhydride)), polypropylfumerates, polyamides (e.g.
polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g. poly(f3-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG
copolymers, and poly(ethyleneimine), poly(ethylene imine)-PEG copolymers.
In some embodiments, polymers in accordance with the present invention include polymers which have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. 177.2600, including but not limited to polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));
polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.
In some embodiments, polymers can be hydrophilic. For example, polymers may comprise anionic groups (e.g., phosphate group, sulphate group, carboxylate group); cationic groups (e.g., quaternary amine group); or polar groups (e.g., hydroxyl group, thiol group, amine group). In some embodiments, a synthetic nanocarrier comprising a hydrophilic polymeric matrix generates a hydrophilic environment within the synthetic nanocarrier. In some embodiments, polymers can be hydrophobic. In some embodiments, a synthetic nanocarrier comprising a hydrophobic polymeric matrix generates a hydrophobic environment within the synthetic nanocarrier. Selection of the hydrophilicity or hydrophobicity of the polymer may have an impact on the nature of materials that are incorporated (e.g., attached) within the synthetic nanocarrier.
In some embodiments, polymers may be modified with one or more moieties and/or functional groups. A variety of moieties or functional groups can be used in accordance with the present invention. In some embodiments, polymers may be modified with polyethylene

- 26 -glycol (PEG), with a carbohydrate, and/or with acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certain embodiments may be made using the general teachings of US Patent No. 5543158 to Gref et al., or WO
publication W02009/051837 by Von Andrian et al.
In some embodiments, polymers may be modified with a lipid or fatty acid group. In some embodiments, a fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. In some embodiments, a fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.
In some embodiments, polymers may be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA"; and homopolymers comprising glycolic acid units, referred to herein as "PGA," and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA." In some embodiments, exemplary polyesters include, for example, polyhydroxyacids; PEG copolymers and copolymers of lactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers, and derivatives thereof. In some embodiments, polyesters include, for example, poly(caprolactone), poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly [ci-(4-aminobuty1)-L-glycolic acid], and derivatives thereof.
In some embodiments, a polymer may be PLGA. PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid. The degradation rate of PLGA can be adjusted by altering the lactic acid:glycolic acid ratio. In some embodiments, PLGA to be used in accordance with the present invention is characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.
In some embodiments, polymers may be one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl

- 27 -methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers.
The acrylic polymer may comprise fully-polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.
In some embodiments, polymers can be cationic polymers. In general, cationic polymers are able to condense and/or protect negatively charged strands of nucleic acids.
Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene imine) (PEI;
Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372) are positively-charged at physiological pH, form ion pairs with nucleic acids.
In embodiments, the synthetic nanocarriers may not comprise (or may exclude) cationic polymers.
In some embodiments, polymers can be degradable polyesters bearing cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J.
Am. Chem.
Soc., 115:11010; Kwon et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am.
Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules, 23:3399).
Examples of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am.
Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633).
The properties of these and other polymers and methods for preparing them are well known in the art (see, for example, U.S. Patents 6,123,727; 5,804,178;
5,770,417; 5,736,372;
5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665;
5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem.
Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem.
Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, a variety of methods for synthesizing certain suitable polymers are

- 28 -described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S. Patents 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
In some embodiments, polymers can be linear or branched polymers. In some embodiments, polymers can be dendrimers. In some embodiments, polymers can be substantially cross-linked to one another. In some embodiments, polymers can be substantially free of cross-links. In some embodiments, polymers can be used in accordance with the present invention without undergoing a cross-linking step. It is further to be understood that the synthetic nanocarriers may comprise block copolymers, graft copolymers, blends, mixtures, and/or adducts of any of the foregoing and other polymers.
Those skilled in the art will recognize that the polymers listed herein represent an exemplary, not comprehensive, list of polymers that can be of use in accordance with the present invention.
In some embodiments, synthetic nanocarriers do not comprise a polymeric component. In some embodiments, synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc. In some embodiments, a non-polymeric synthetic nanocarrier is an aggregate of non-polymeric components, such as an aggregate of metal atoms (e.g., gold atoms).
The doses or dosage forms according to the invention can comprise pharmaceutically acceptable excipients, such as preservatives, buffers, saline, or phosphate buffered saline.
The compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at useful dosage forms. In some embodiments, the compositions of the dosings are suspended in sterile saline solution for injection together with a preservative. In some embodiments, synthetic nanocarriers are suspended in sterile saline solution for injection together with a preservative.
In embodiments, when preparing synthetic nanocarriers for use with immunosuppressants, methods for attaching components to the synthetic nanocarriers may be useful. If the component is a small molecule it may be of advantage to attach the component to a polymer prior to the assembly of the synthetic nanocarriers. In embodiments, it may also be an advantage to prepare the synthetic nanocarriers with surface groups that are used to attach the component to the synthetic nanocarrier through the use of these surface groups

- 29 -rather than attaching the component to a polymer and then using this polymer conjugate in the construction of synthetic nanocarriers.
In certain embodiments, the attaching can be with a covalent linker. In embodiments, components according to the invention can be covalently attached to the external surface via a 1,2,3-triazole linker formed by the 1,3-dipolar cycloaddition reaction of azido groups on the surface of the nanocarrier with a component containing an alkyne group or by the 1,3-dipolar cycloaddition reaction of alkynes on the surface of the nanocarrier with a component containing an azido group. Such cycloaddition reactions are preferably performed in the presence of a Cu(I) catalyst along with a suitable Cu(I)-ligand and a reducing agent to reduce Cu(II) compound to catalytic active Cu(I) compound. This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be referred as the click reaction.
Additionally, the covalent attaching may comprise a covalent linker that comprises an amide linker, a disulfide linker, a thioether linker, a hydrazone linker, a hydrazide linker, an imine or oxime linker, an urea or thiourea linker, an amidine linker, an amine linker, and a sulfonamide linker.
An amide linker is formed via an amide bond between an amine on one component such as an immunosuppressant with the carboxylic acid group of a second component such as the nanocarrier. The amide bond in the linker can be made using any of the conventional amide bond forming reactions with suitably protected amino acids and activated carboxylic acid such N-hydroxysuccinimide-activated ester.
A disulfide linker is made via the formation of a disulfide (S-S) bond between two sulfur atoms of the form, for instance, of R1-S-S-R2. A disulfide bond can be formed by thiol exchange of a component containing thiol/mercaptan group(-SH) with another activated thiol group on a polymer or nanocarrier or a nanocarrier containing thiol/mercaptan groups with a component containing activated thiol group.

N -N
I

A triazole linker, specifically a 1,2,3-triazole of the form R 2 , wherein R1 and R2 may be any chemical entities, is made by the 1,3-dipolar cycloaddition reaction of an azide attached to a first component, such as the nanocarrier, with a terminal alkyne attached to a second component, such as the immunosuppressant. The 1,3-dipolar cycloaddition reaction is performed with or without a catalyst, preferably with Cu(I)-catalyst, which links the two components through a 1,2,3-triazole function. This chemistry is described in detail by

- 30 -Sharples s et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) and Meldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and is often referred to as a "click" reaction or CuAAC.
In embodiments, a polymer containing an azide or alkyne group, terminal to the polymer chain is prepared. This polymer is then used to prepare a synthetic nanocarrier in such a manner that a plurality of the alkyne or azide groups are positioned on the surface of that nanocarrier. Alternatively, the synthetic nanocarrier can be prepared by another route, and subsequently functionalized with alkyne or azide groups. The component is prepared with the presence of either an alkyne (if the polymer contains an azide) or an azide (if the polymer contains an alkyne) group. The component is then allowed to react with the nanocarrier via the 1,3-dipolar cycloaddition reaction with or without a catalyst which covalently attaches the component to the particle through the 1,4-disubstituted 1,2,3-triazole linker.
A thioether linker is made by the formation of a sulfur-carbon (thioether) bond in the form, for instance, of R1-S-R2. Thioether can be made by either alkylation of a thiol/mercaptan (-SH) group on one component with an alkylating group such as halide or epoxide on a second component. Thioether linkers can also be formed by Michael addition of a thiol/mercaptan group on one component to an electron-deficient alkene group on a second component containing a maleimide group or vinyl sulfone group as the Michael acceptor. In another way, thioether linkers can be prepared by the radical thiol-ene reaction of a thiol/mercaptan group on one component with an alkene group on a second component.
A hydrazone linker is made by the reaction of a hydrazide group on one component with an aldehyde/ketone group on the second component.
A hydrazide linker is formed by the reaction of a hydrazine group on one component with a carboxylic acid group on the second component. Such reaction is generally performed using chemistry similar to the formation of amide bond where the carboxylic acid is activated with an activating reagent.
An imine or oxime linker is formed by the reaction of an amine or N-alkoxyamine (or aminooxy) group on one component with an aldehyde or ketone group on the second component.
An urea or thiourea linker is prepared by the reaction of an amine group on one component with an isocyanate or thioisocyanate group on the second component.
An amidine linker is prepared by the reaction of an amine group on one component with an imidoester group on the second component.

-31 -An amine linker is made by the alkylation reaction of an amine group on one component with an alkylating group such as halide, epoxide, or sulfonate ester group on the second component. Alternatively, an amine linker can also be made by reductive amination of an amine group on one component with an aldehyde or ketone group on the second component with a suitable reducing reagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.
A sulfonamide linker is made by the reaction of an amine group on one component with a sulfonyl halide (such as sulfonyl chloride) group on the second component.
A sulfone linker is made by Michael addition of a nucleophile to a vinyl sulfone.
Either the vinyl sulfone or the nucleophile may be on the surface of the nanocarrier or attached to a component.
The component, preferably an immunosuppressant, can also be conjugated to the nanocarrier via non-covalent conjugation methods. For example, a negative charged immunosuppressant can be conjugated to a positive charged nanocarrier through electrostatic adsorption. A component containing a metal ligand can also be conjugated to a nanocarrier containing a metal complex via a metal-ligand complex.
In embodiments, the component can be attached to a polymer, for example polylactic acid-block-polyethylene glycol, prior to the assembly of the synthetic nanocarrier or the synthetic nanocarrier can be formed with reactive or activatible groups on its surface. In the latter case, the component may be prepared with a group which is compatible with the attachment chemistry that is presented by the synthetic nanocarriers' surface.
In other embodiments, a peptide component can be attached to VLPs or liposomes using a suitable linker. A linker is a compound or reagent that is capable of attaching two molecules together.
In an embodiment, the linker can be a homobifuntional or heterobifunctional reagent as described in Hermanson 2008. For example, an VLP or liposome synthetic nanocarrier containing a carboxylic group on the surface can be treated with a homobifunctional linker, adipic dihydrazide (ADH), in the presence of EDC to form the corresponding synthetic nanocarrier with the ADH linker. The resulting ADH linked synthetic nanocarrier is then conjugated with a peptide component containing an acid group via the other end of the ADH
linker on nanocarrier to produce the corresponding VLP or liposome peptide conjugate.
For detailed descriptions of available conjugation methods, see Hermanson G T
"Bioconjugate Techniques", 2nd Edition Published by Academic Press, Inc., 2008. In addition to covalent attachment the component can be attached by adsorption to a pre-formed

- 32 -synthetic nanocarrier or it can be attached by encapsulation during the formation of the synthetic nanocarrier.
Any immunosuppressant as provided herein can be used and attached to the synthetic nanocarriers. Immunosuppressants include, but are not limited to, statins;
mTOR inhibitors, such as rapamycin or a rapamycin analog; TGF-P signaling agents; TGF-P
receptor agonists;
histone deacetylase (HDAC) inhibitors; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-i3 inhibitors; adenosine receptor agonists;
prostaglandin E2 agonists; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor; proteasome inhibitors; kinase inhibitors; G-protein coupled receptor agonists; G-protein coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors and oxidized ATPs.
Immunosuppressants also include IDO, vitamin D3, cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine, 6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide, interleukins (e.g., IL-1, IL-10), cyclosporine A, siRNAs targeting cytokines or cytokine receptors and the like.
Examples of statins include atorvastatin (LIPITOR , TORVAST ), cerivastatin, fluvastatin (LESCOL , LESCOL XL), lovastatin (MEVACOR , ALTOCOR , ALTOPREV), mevastatin (COMPACTIN ), pitavastatin (LIVALO , PTA VA ), rosuvastatin (PRAVACHOL , SELEKTINE , LIPOSTAr), rosuvastatin (CRESTOR ), and simvastatin (ZOCOR , LIPEX ).
Examples of mTOR inhibitors include rapamycin and analogs thereof (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap), C16-(S)-butylsulfonamidorapamycin (C16-BSrap), C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry & Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available from Selleck, Houston, TX, USA).
Examples of TGF-P signaling agents include TGF-P ligands (e.g., activin A, GDF1, GDF11, bone morphogenic proteins, nodal, TGF-Ps) and their receptors (e.g., ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B, TGFPRI, TGFPRII), R-

- 33 -SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD8), and ligand inhibitors (e.g., follistatin, noggin, chordin, DAN, lefty, LTBP 1, THBS1, Decorin).
Examples of inhibitors of mitochondrial function include atractyloside (dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanide m-chlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylis gurnrnifera), CGP-37157, (-)-Deguelin (e.g., from Mundulea sericea), F16, hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360, SFK1, and valinomycin (e.g., from Streptornyces fulvissirnus) (EMD4Biosciences, USA).
Examples of P38 inhibitors include SB-203580 (4-(4-Fluoropheny1)-2-(4-methylsulfinylpheny1)-5-(4-pyridy1)1H-imidazole), SB-239063 (trans-1-(4hydroxycyclohexyl)-4-(fluoropheny1)-5-(2-methoxy-pyrimidin-4-y1) imidazole), SB-220025 (5-(2amino-4-pyrimidiny1)-4-(4-fluoropheny1)-1-(4-piperidinyl)imidazole)), and ARRY-797.
Examples of NF (e.g., NK-i3) inhibitors include IFRD1, 2-(1,8-naphthyridin-2-y1)-Phenol, 5-aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE (Caffeic Acid Phenethylester), diethylmaleate, IKK-2 Inhibitor IV, IMD 0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CH0], NFKB Activation Inhibitor III, NF-KB Activation Inhibitor II, JSH-23, parthenolide, Phenylarsine Oxide (PAO), PPM-18, pyrrolidinedithiocarbamic acid ammonium salt, QNZ, RO 106-9920, rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamide J, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490), and wedelolactone.
Examples of adenosine receptor agonists include CGS-21680 and ATL-146e.
Examples of prostaglandin E2 agonists include E-Prostanoid 2 and E-Prostanoid 4.
Examples of phosphodiesterase inhibitors (non-selective and selective inhibitors) include caffeine, aminophylline, IBMX (3-isobuty1-1-methylxanthine), paraxanthine, pentoxifylline, theobromine, theophylline, methylated xanthines, vinpocetine, EHNA
(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFANTm), milrinone, levosimendon, mesembrine, ibudilast, piclamilast, luteolin, drotaverine, roflumilast (DAXAS TM, DALIRESPTm), sildenafil (REVATION , VIAGRA ), tadalafil (ADCIRCA , .. CIALIS ), vardenafil (LEVITRA , STAXYN ), udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolo pyrimidin-7-1.
Examples of proteasome inhibitors include bortezomib, disulfiram, epigallocatechin-3-gallate, and salinosporamide A.

- 34 -Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab (ERBITUX ), imatinib (GLEEVEC ), trastuzumab (HERCEPTIN ), gefitinib (IRESSA
), ranibizumab (LUCENTIS ), pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080, pazopanib, and mubritinib.
Examples of glucocorticoids include hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), and aldosterone.
Examples of retinoids include retinol, retinal, tretinoin (retinoic acid, RETIN-A ), isotretinoin (ACCUTANE , AMNESTEEM , CLARAVIS , SOTRET ), alitretinoin (PANRETIN ), etretinate (TEGISONTm) and its metabolite acitretin (SORIATANE ), tazarotene (TAZORAC , AVAGE , ZORAC ), bexarotene (TARGRETIN ), and adapalene (DIFFERIN ).
Examples of cytokine inhibitors include IL lra, IL1 receptor antagonist, IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin, Cyclosporin A, Pentamidine, and Pentoxifylline (PENTOPAK , PENTOXIL , TRENTAL ).
Examples of peroxisome proliferator-activated receptor antagonists include GW9662, PPARy antagonist III, G335, and T0070907 (EMD4Biosciences, USA).
Examples of peroxisome proliferator-activated receptor agonists include pioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY 171883, PPARy activator, Fmoc-Leu, troglitazone, and WY-14643 (EMD4Biosciences, USA).
Examples of histone deacetylase inhibitors include hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic tetrapeptides (such as trapoxin B) and depsipeptides, benzamides, electrophilic ketones, aliphatic acid compounds such as phenylbutyrate and valproic acid, hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589), benzamides such as entinostat (MS-275), CI994, and mocetinostat (MGCD0103), nicotinamide, derivatives of NAD, dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.
Examples of calcineurin inhibitors include cyclosporine, pimecrolimus, voclosporin, and tacrolimus.
Examples of phosphatase inhibitors include BN82002 hydrochloride, CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin, ethyl-3,4-dephostatin, fostriecin sodium salt, MAZ51, methyl-3,4-dephostatin, NSC 95397, norcantharidin, okadaic acid

- 35 -ammonium salt from prorocentrum concavum, okadaic acid, okadaic acid potassium salt, okadaic acid sodium salt, phenylarsine oxide, various phosphatase inhibitor cocktails, protein phosphatase 1C, protein phosphatase 2A inhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2, and sodium orthovanadate.
D.
METHODS OF MAKING AND USING THE COMPOSITIONS AND RELATED
METHODS
Viral vectors can be made with methods known to those of ordinary skill in the art or as otherwise described herein. For example, viral vectors can be constructed and/or purified using the methods set forth, for example, in U.S. Pat. No. 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).
Viral vectors, such as AAV vectors, may be produced using recombinant methods.
For example, the methods can involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein or fragment thereof; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV
vector into the AAV capsid proteins.
The components to be cultured in the host cell to package a viral vector in a capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant viral vector, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell can contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. The recombinant viral vector, rep sequences, cap sequences, and helper functions for producing the viral vector may be delivered to the packaging host cell using any appropriate genetic element. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a

- 36 -suitable method is not a limitation on the present invention. See, e.g., K.
Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.
In some embodiments, recombinant AAV vectors may be produced using the triple transfection method (e.g., as described in detail in U.S. Pat. No. 6,001,650, the contents of which relating to the triple transfection method are incorporated herein by reference).
Typically, the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (such as comprising a transgene) to be packaged into AAV particles, an AAV
helper function vector, and an accessory function vector. Generally, an AAV
helper function vector encodes AAV helper function sequences (rep and cap), which function in trans for productive AAV replication and encapsulation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV
virions (i.e., AAV virions containing functional rep and cap genes). The accessory function vector can encode nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication. The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA
splicing, AAV
DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus. Other methods for producing viral vectors are known in the art. Moreover, viral vectors are available commercially.
In regard to synthetic nanocarriers attached to immunosuppressants, methods for attaching components to synthetic nanocarriers may be useful. Synthetic nanocarriers may be prepared using a wide variety of methods known in the art. For example, synthetic nanocarriers can be formed by methods such as nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor, conductive, magnetic, organic, and other nanomaterials have been described (Pellegrino et al., 2005, Small, 1:48;
Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 13:3843).
Additional methods have been described in the literature (see, e.g., Doubrow, Ed., "Microcapsules and

- 37 -Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992;
Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; US Patents 5578325 and 6007845; P.
Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5(6):843-853 (2010)).
Various materials may be encapsulated into synthetic nanocarriers as desirable using a variety of methods including but not limited to C. Astete et al., "Synthesis and characterization of PLGA nanoparticles" J. Biomater. Sci. Polymer Edn, Vol.
17, No. 3, pp.
247-289 (2006); K. Avgoustakis "Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery" Current Drug Delivery 1:321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles" Nanomedicine 2:8¨
21(2006); P.
Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5(6):843-853 (2010). Other methods suitable for encapsulating materials into synthetic nanocarriers may be used, including without limitation methods disclosed in United States Patent 6,632,671 to Unger issued October 14, 2003.
In certain embodiments, synthetic nanocarriers are prepared by a nanoprecipitation process or spray drying. Conditions used in preparing synthetic nanocarriers may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, "stickiness," shape, etc.). The method of preparing the synthetic nanocarriers and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may depend on the materials to be attached to the synthetic nanocarriers and/or the composition of the polymer matrix.
If synthetic nanocarriers prepared by any of the above methods have a size range outside of the desired range, such synthetic nanocarriers can be sized, for example, using a sieve.
Elements (i.e., components) of the synthetic nanocarriers may be attached to the overall synthetic nanocarrier, e.g., by one or more covalent bonds, or may be attached by means of one or more linkers. Additional methods of functionalizing synthetic nanocarriers may be adapted from Published US Patent Application 2006/0002852 to Saltzman et al., Published US Patent Application 2009/0028910 to DeSimone et al., or Published International Patent Application WO/2008/127532 Al to Murthy et al.

- 38 -Alternatively or additionally, synthetic nanocarriers can be attached to components directly or indirectly via non-covalent interactions. In non-covalent embodiments, the non-covalent coupling is mediated by non-covalent interactions including but not limited to charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. Such couplings may be arranged to be on an external surface or an internal surface of a synthetic nanocarrier.
In embodiments, encapsulation and/or absorption is a form of coupling.
Compositions provided herein may comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH
adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxycholate), solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol).
Compositions according to the invention may comprise pharmaceutically acceptable excipients. The compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at useful dosage forms.
Techniques suitable for use in practicing the present invention may be found in Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill Livingstone. In an embodiment, compositions are in a sterile saline solution for injection together with a preservative.
It is to be understood that the compositions of the invention can be made in any suitable manner, and the invention is in no way limited to compositions that can be produced using the methods described herein. Selection of an appropriate method of manufacture may require attention to the properties of the particular moieties being associated.

- 39 -In some embodiments, compositions are manufactured under sterile conditions or are terminally sterilized. This can ensure that resulting compositions are sterile and non-infectious, thus improving safety when compared to non-sterile compositions.
This provides a valuable safety measure, especially when subjects receiving the compositions have immune defects, are suffering from infection, and/or are susceptible to infection. In some embodiments, the compositions may be lyophilized and stored in suspension or as lyophilized powder depending on the formulation strategy for extended periods without losing activity.
Administration according to the present invention may be by a variety of routes, including but not limited to subcutaneous, intravenous, or intraperitoneal routes. The compositions referred to herein may be manufactured and prepared for administration, such as concomitant administration, using conventional methods.
The compositions of the invention can be administered in effective amounts, such as the effective amounts described elsewhere herein. Doses of dosage forms may contain varying amounts of immunosuppressants, according to the invention. Doses of dosage forms may contain varying amounts of viral vectors, according to the invention. The amount of respective components present in the dosage forms can be varied according to the nature of the components, the therapeutic benefit to be accomplished, and other such parameters. In embodiments, dose ranging studies can be conducted to establish optimal therapeutic amounts of the components to be present in the dosage forms. In embodiments, the components are present in the dosage forms in an amount effective to reduce an undesired immune (e.g., humoral) response to the viral vector and/or result in efficacious and/or durable and/or comparable expression upon administration to a subject. It may be possible to determine amounts of the components effective to reduce an undesired immune response using conventional dose ranging studies and techniques in subjects. Dosage forms may be administered at a variety of frequencies (i.e., according to an administration schedule).
Another aspect of the disclosure relates to kits. In some embodiments, the kit comprises one or more doses of viral vectors and/or one or more doses synthetic nanocarriers attached to an immunosuppressant, as provided herein. Each of the doses of a kit can be contained within separate containers or within the same container in the kit.
In some embodiments, the container is a vial or an ampoule. In some embodiments, each of the doses can be contained within a solution separate from the container, such that the dose may be added to a container at a subsequent time. In some embodiments, the doses are in lyophilized form each in a separate container or in the same container, such that they may be

- 40 -reconstituted at a subsequent time. In some embodiments, the kit further comprises instructions for reconstitution, mixing, administration, etc. In some embodiments, the instructions include a description of the methods described herein.
Instructions can be in any suitable form, e.g., as a printed insert or a label. In some embodiments, the kit further comprises one or more syringes.
An administration schedule can be determined by varying the number of dosing(s) and/or the length of time between the dosing(s) and assessing an undesired immune response to a viral vector and/or expression of a transgene or nucleic acid material thereof. For example, after administering dosing(s) of the viral vector and/or dosing(s) of the synthetic nanocarriers attached to an immunosuppressant transgene expression and/or an undesired immune response to a viral vector can be measured. This undesired immune response and/or expression can be compared to the same type of immune response and/or expression that occurs without the dosing(s) of the synthetic nanocarriers attached to an immunosuppressant, such as when the dosings of viral vector has occurred without concomitant administration with synthetic nanocarriers attached to an immunosuppressant as provided herein.
Administration schedules may be determined by starting with a test schedule and using known scaling techniques (such as allometric or isometric scaling) as appropriate. In another embodiment, the administration schedule may be determined by testing various schedules in a subject, e.g., through direct experimentation based on experience and guiding data.
EXAMPLES
Example 1: Synthesis of Synthetic Nanocarriers Comprising an Immunosuppressant Synthetic nanocarriers (e.g., PLA and/or PLA-PEG nanocarriers) comprising (e.g., encapsulating) an immunosuppressant, for example rapamycin; (e.g., ImmTOR) were produced. Preferably, in some embodiments of any one of the methods or compositions provided herein the synthetic nanocarriers comprising an immunosuppressant are produced by any one of the methods of US Publication No. US 2016/0128986 Al and US
Publication No. US 2016/0128987 Al, the described methods of such production and the resulting synthetic nanocarriers being incorporated herein by reference in their entirety. In any one of the methods or compositions provided herein, the synthetic nanocarriers comprising an immunosuppressant, such as rapamycin, are such incorporated synthetic nanocarriers.

- 41 -Example 2: Systemic Immunogenicity Considerations for Adeno-Associated Virus (AAV)-Mediated Gene Therapy AAV immunogenicity and AAV toxicity represent two major challenges for the gene therapy field, and in many cases are inextricably linked. Immunogenicity of AAV vectors is thought to cause or exacerbate many of the adverse events associated with AAV
gene therapy, such as hepatotoxicity and thrombotic microangiopathy. Moreover, these adverse events tend to be correlated with vector dose, increasing in both prevalence and severity with higher doses. Not surprisingly, higher vector doses are also associated with increased immunogenicity, leading to a vicious circle when vector doses of 1E14 vg/kg or higher are required for efficacy in neuromuscular diseases. These issues will likely require a multipronged approach to improve vector transduction efficiency and mitigate vector immunogenicity, and perhaps a shift in the 'one-and-done' paradigm of gene therapy.
Immunogenicity of AAV gene therapy is much more complex and nuanced than that for most protein biologic therapies, which are typically affected only by anti-drug antibodies (ADAs). ADAs also limit systemic AAV gene therapies, through pre-existing anti-AAV
neutralizing antibodies that occur through natural exposure to wildtype AAV
and which limit patient eligibility for gene therapy, and through de novo formation of neutralizing ADAs that occur after treatment and prevent the ability to re-dose AAV gene therapies.
However, many of the toxicities of AAV gene therapies appear to be driven by innate immune responses and adaptive CD8 T cell responses.
Early clinical trials in hemophilia reported delayed elevation in serum levels of liver transaminases, which is indicative of liver inflammation and hepatoxicity. In some patients, the increase in liver enzymes correlated with a decrease in transgene expression and the appearance of circulating capsid-specific CD8 T cells. In many cases, the elevated liver .. transaminase levels and further loss of transgene expression could be successfully treated with steroids. The induction of an adaptive immune response indicates the activation of innate antigen-presenting cells, such as dendritic cells or macrophages. APCs express toll-like receptors (TLR) which are involved in recognition of pathogen-associated molecular patterns. The DNA payload of AAV vectors may contain CpG motifs that bind and activate TLR9, an innate immune sensor for unmethylated CpG dinucleotides. Similarly, the capsid itself can be recognized by TLR2 leading to activation of the APCs which can lead to the induction of adaptive immune responses. A curious aspect of the hepatoxicity associated with AAV gene therapy is the delayed onset, typically 4-8 weeks after dosing.
This delay

- 42 -suggests that AAV capsid antigens persists in some tissues and that some secondary event, or possibly cumulative hepatic stress, results in the activation of CD8 T cells.
Adverse events related to elevated liver transaminases have now been widely reported in AAV gene therapy clinical trials, with increased prevalence at higher vector doses. AAV
gene therapy for neuromuscular diseases have typically required doses in excess of 1E14 vg/kg. Onasemnogene abeparvovec, an AAV9 therapy for spinal muscular atrophy (SMA), and the first systemic AAV gene therapy approved by the FDA, has been administered to over 800 patients. Approximately one third of the patients receiving a dose of 1.1E14 vg/kg have experienced at least one adverse event of hepatotoxicity. Three patients have been reported to experience severe hepatoxicity 7-8 weeks after treatment. Liver biopsies indicated hepatocyte degeneration and inflammatory infiltrates comprised primarily of CD8 T
cells. All three patients recovered after treatment with methylprednisolone.
However, tragically, three patients with X-linked myotubular myopathy (XLMTM) died 20-40 weeks after receiving a vector dose of 3E14 vg/kg. The patients presented with signs of severe hepatotoxicity, with bilirubin levels 35-60 times the upper limit of normal and delayed elevation in AST and ALT, and evidence of cholestasis, periportal and bile ductal reaction, and secondary fibrosis at autopsy. Reports indicating a lack of inflammatory cellular infiltrates and ineffective treatment with steroids and immunosuppressants, suggest that the toxicity was not immune-related. All three patients showed pre-existing evidence of hyperbilirubinemia and intrahepatic cholestasis. However, an early innate immune response to the massive AAV dose could have contributed to hepatic stress resulting in exacerbation of the underlying disease. The clinical trial was halted and eventually restarted at a lower dose of 1E14 vg/kg. Unfortunately, a fourth patient died after treatment with the lower dose.
High vector doses of AAV have also been associated with thrombotic microangiopathy (TMA) or atypical hemolytic uremic syndrome (aHUS), a syndrome characterized by hemolytic anemia, thrombocytopenia, and acute kidney injury, and associated with complement activation. Complement is a key component of the innate immune system and is comprised of plasma proteins that undergo a complex cascade of biochemical reactions that can result in direct killing of pathogens or opsonizing them for clearance, as well as chemoattraction and activation of innate immune cells and the release of vasoactive mediators. AAV can activate complement through binding of antibodies that fix complement (classical pathway) or direct binding of complement components (alternative pathway). In SMA, three cases of TMA have been reported. The overall incidence is low,

-43 -with over 800 patients treated; nonetheless, one of the affected subjects has had complications with persistent hypertension while another took three months to resolve. In DMD, two small clinical trials involving two different AAV product candidates reported four cases of severe adverse reactions involving aHUS out of 15 total patients dosed (Mendell Mol Ther 2021). One case occurred at a dose of 5E13 vg/kg, while the other three occurred at vector doses of 1-3E14 vg/kg. An amendment was made to the Phase 3 clinical trial in DMD
to exclude patients with certain mutations as a result of three cases of myocarditis. More tragically, a patient death in the Phase lb trial was reported. No further details were available, and the clinical trial was put on hold.
The different manifestations and relative incidence of adverse reactions suggest that multiple variables may contribute to the overall risk, such as vector design, vector manufacturing and quality attributes, underlying disease, and co-morbidities.
Retrospective analysis of hemophilia B gene therapy trials suggest that higher CpG content correlated with higher incidence of liver enzyme elevation, stronger CD8 T cell responses and less durable transgene expression. In DMD, a third clinical program, has yet to announce a serious adverse event related to aHUS, with 77 patients treated. The basis for a high incidence of aHUS in two programs but not a third is unknown. It is conceivable that different capsids would have different profiles with respect to fixation of complement or binding to pre-existing antibodies. One major unknown with all AAV clinical trials is the degree of impurities from the manufacturing process, such as percentage of empty capsids, or host cell impurities. Since AAV doses are calculated based on determination of vector genomes (or 'full' capsids), the ratio of empty:full capsids could have a huge bearing on the total capsid dose.
For all the notable successes and continued promise of the AAV field, there is still tremendous potential to improve the safety and efficacy of recombinant AAV
vectors. A
capsid dose of 1E14 vg/kg represents a dose of over a quadrillion viral particles (not including empty capsids) in a young child, which is 10-30-greater than the total number of cells in the body. Increasing the ratio of full:empty capsids and reducing CpG
content provide an immediately actionable strategy to improve safety. Another potential strategy to increase transduction efficiency and reduce CD8 T cell responses is to introduce capsid mutations that minimize processing by the proteasome, which results in capsid degradation and presentation of immunogenic epitopes on MHC class I molecules. Longer term strategies include sophisticated capsid engineering approaches to design or evolve capsids that show

- 44 -better targeting of specific tissues, such as muscle. Improvements in promoter and enhancer design could also improve efficacy at lower doses. In addition, engineering of more efficient transgenes, as exemplified by the Padua variant used in hemophilia B, could allow for lower vector doses. Ultimately, a breakthrough in radically improving transduction efficiency may require a better understanding of the key rate limiting steps of AAV
transduction, including transit through the cell from the endosome to the nucleus, viral uncoating, maintenance as a stable episome, and interaction with host cell factors.
Both capsid engineering and pharmacological strategies are being pursued to mitigate the immunogenicity of AAV capsids. Some strategies, such as mutagenesis of capsids to remove antibody epitopes or use of plasmapheresis or IgG protease to eliminate anti-AAV
antibodies, only address the pre-existing antibody component, without addressing innate immunity or T cell responses. Indeed, the very presence of pre-existing anti-AAV antibodies is indicative of prior exposure to AAV and a high likelihood of the presence of AAV-specific memory T and B cells. High vector doses, which can overcome low levels of neutralizing antibodies, has enabled some groups to loosen the threshold antibody titer for inclusion into clinical trials, even though the pre-existing memory T cells and complement fixation by pre-existing low titer antibodies could still potentially exacerbate the toxicities observed with high vector doses. Combination immunosuppressive regimens, such as rapamycin and corticosteroids or rapamycin and rituximab are being evaluated clinically for mitigation of immunogenicity. Rapamycin is an inhibitor of the mTOR pathway, which dosed chronically, can inhibit effector T cell activation. Rapamycin encapsulated into nanoparticles (also referred to as ImmTOR in some embodiments) is also being evaluated for the induction of antigen-specific immune tolerance to AAV vectors. A dose of ImmTOR
nanoparticles administered at the time of AAV gene therapy treatment has been shown to inhibit T and B
cell responses to AAV and enable vector redosing. ImmTOR has also been shown to have hepatoprotective properties in animal models of liver inflammation. Notably rapamycin has also been shown to enhance AAV vector transduction through induction of autophagy.
The stakes are high for mitigating the toxicity of high vector doses. The benefit of AAV gene therapy has been undeniable in SMA and highly promising in DMD. Even in XLMTM, those patients that tolerated therapy showed remarkable improvement.
Further improvement might be achievable if the therapeutic window could be safely widened. The ultimate goal should be to provide similar efficacy at lower vector doses.
This may require a combination of vector engineering and pharmacological strategies to increase vector

- 45 -transduction efficiency and mitigate immunogenicity. AAV gene therapy has been historically viewed as 'one-and-done', meaning that a single dose of therapy is expected to provide years, if not a lifetime, of therapeutic benefit. This notion has been challenged by the waning of efficacy in a hemophilia A gene therapy trial and the loss of efficacy observed in patients that experience hepatotoxicity. If the barrier to vector redosing, currently limited by the formation of high titers of neutralizing antibodies after initial exposure, can be overcome, it not only opens the possibility to restore therapeutic benefit in patients that have experienced loss of transgene expression, but may also provide a path to treatment, such as of neuromuscular diseases, by allowing for multiple smaller doses of gene therapy rather than a .. single high dose of 1-3E14vg/kg. The potential of providing a therapeutic outcome by delivering multiple lower doses over a finite period of time (e.g., 1- 2 weeks or 1-3 months) may be the next gene therapy paradigm that facilitates the next wave of approved gene therapy drugs with lower toxicity profiles for unmet orphan diseases - "one and done" may be traded in for "lower and slower".

Claims

What is claimed is:

1. A method comprising:
a dosing comprising, (i) one or more doses of a viral vector, such as an AAV vector, that is not attached to any synthetic nanocarriers, wherein the dose(s) of the viral vector are each a lower dose, and (ii) at least one dose of synthetic nanocarriers that are attached to an immunosuppressant, such as rapamycin, and that comprise no viral vector antigens of the viral vector;
wherein the lower dose is lower than a dose of the viral vector administered without concomitant administration of the synthetic nanocarriers that are attached to an immunosuppressant but results in comparable transgene expression as the dose of the viral vector administered without concomitant administration of the synthetic nanocarriers that are attached to an immunosuppressant, optionally, according to an administration schedule that reduces an undesired immune (eg., humoral) response to the viral vector and/or results in efficacious transgene or nucleic acid material expression and/or provides durable transgene or nucleic acid material expression and/or results in comparable transgene expression.

2. The method of claim 1, wherein (i) and (ii) of the dosing are concomitantly administered.

3. The method of claim 1 or 2, wherein the dosing comprising (i) and (ii) is repeated, optionally, within 70 days, 2 months or 1 month of a previous dosing of (i) and (ii).

4. The method of any one of the preceding claims, wherein the method further comprises assessing the undesired immune response and/or transgene or nucleic acid material expression in the subject prior to and/or after the administration of the administration(s).

5. The method of any one of the preceding claims, wherein the method further comprises identifying the subject as having or at risk of having an undesired immune response to the viral vector and/or as being in need of effective or durable transgene or nucleic acid material expression.

6. A composition comprising:
(i) more than one dose of a viral vector, such as an AAV vector, that is not attached to any synthetic nanocarriers, wherein the doses of the viral vector are each lower doses, and/or (ii) at least one dose of synthetic nanocarriers that are attached to an immunosuppressant, such as rapamycin, and that comprise no viral vector antigens of the viral vector;
wherein the lower dose is lower than a dose of the viral vector administered without concomitant administration of the synthetic nanocarriers that are attached to an immunosuppressant but results in comparable transgene expression as the dose of the viral vector administered without concomitant administration of the synthetic nanocarriers that are attached to an immunosuppressant, optionally, for use in a method of reducing an undesired immune (eg., humoral) response to the viral vector and/or resulting in efficacious and/or durable transgene or nucleic acid material expression and/or resulting in comparable transgene expression.

7. The method or composition of any one of the preceding claims, wherein the method further comprises determining a protocol or administration schedule for (i) and (ii).

8. The method or composition of any one of the preceding claims, wherein the method is any method as provided herein.

9. The composition of any one of claims 6-8, wherein the composition is a kit and the doses are each housed in a container in the kit.

10. The composition of any one of claims 6-9, wherein the composition further comprises a pharmaceutically acceptable carrier.

11. The method or composition of any of the preceding claims, wherein the immunosuppressants comprise a statin, an mTOR inhibitor, a TGF-0 signaling agent, a corticosteroid, an inhibitor of mitochondrial function, a P38 inhibitor, an NF-KB inhibitor, an adenosine receptor agonist, a prostaglandin E2 agonist, a phosphodiesterasse 4 inhibitor, an HDAC inhibitor or a proteasome inhibitor.

12. The method or composition of claim 11, wherein the immunosuppressant is an mTOR
inhibitor.

13. The method or composition of claim 12, wherein the mTOR inhibitor is rapamycin or a rapamycin analog.

14. The method or composition of any one of the preceding claims, wherein the viral vector is an AAV vector, such as an AAV8 vector.

15. The method or composition of any of the preceding claims, wherein a load of the immunosuppressant is on average across the population of synthetic nanocarriers is between 0.1% and 50%.

16. The method or composition of claim 15, wherein the load of the immunosuppressant on average across the population of synthetic nanocarriers is between 1% and 30%, 1% and 25%, 1% and 20%, 4% and 30%, 4% and 25%, 4% and 20%, 8% and 30%, 8% and 25%, or 8% and 20%.

17. The method or composition of any of the preceding claims, wherein the synthetic nanocarriers are polymeric.

18. The method or composition of claim 17, wherein the polymeric nanocarriers comprise polymer that is a non-methoxy-terminated, pluronic polymer.

19. The method or composition of claim 17 or 18, wherein the polymeric nanocarriers comprise a polyester, a polyester coupled to a polyether, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine.

20. The method or composition of claim 19, wherein the polyester comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone.

21. The method or composition of claim 19 or 20, wherein the polymeric nanocarriers comprise a polyester and a polyester coupled to a polyether.

22. The method or composition of any of claims 19-21, wherein the polyether comprises polyethylene glycol or polypropylene glycol.

23. The method or composition of any of the preceding claims, wherein the mean of a particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers of the population is a diameter greater than 100nm.

24. The method or composition of claim 23, wherein the diameter is greater than 150nm.

25. The method or composition of claim 24, wherein the diameter is greater than 200nm.

26. The method or composition of claim 25, wherein the diameter is greater than 250nm.

27. The method or composition of claim 26, wherein the diameter is greater than 300nm.

28. The method or composition of any one of claims 23-27, where the diameter is less than 500nm.

29. The method or composition of any one of claims 23-27, where the diameter is less than 450nm.

30. The method or composition of any one of claims 23-27, where the diameter is less than 400nm.

31. The method or composition of any one of claims 23-27, where the diameter is less than 350nm.

32. The method or composition of any one of claims 23-26, where the diameter is less than 300nm.

33. The method or composition of any one of claims 23-25, where the diameter is less than 250nm.

34. The method or composition of claim 23 or 24, where the diameter is less than 200nm.

35. The method or composition of any of the preceding claims, wherein an aspect ratio of the synthetic nanocarriers of the population is greater than or equal to 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

36. The method or composition of any one of the preceding claims, wherein each lower dose of the viral vector is less than 1e1014 vector genomes/kg (e.g., at least 1/5, no more than 1/5 or 1/5 of 1e1014 vector genomes/kg) or wherein each lower dose is less than 5e13 vector genomes/kg (e.g., at least 1/5, no more than 1/5 or 1/5 of 5e13 vector genomes/kg), such as when the subject is human.

37. The method or composition of any one of the preceding claims, wherein when (i) of the dosing(s) comprise more than one dose of a viral vector, the doses of (i) of each dosing are administered over a 1 to 2 week period.

38. The method or composition of any one of the preceding claims, wherein when (i) of the dosing(s) comprise more than one dose of a viral vector, the doses of (i) of each dosing are administered over a 1-3 month period.

39. The method or composition of any one of the preceding claims, wherein (ii) is administered concomitantly with the first dose of (i) for each dosing.

40. The method or composition of any one of the preceding claims, wherein the subject is experiencing or has experienced loss of transgene expression.

41. The method or composition of any one of the preceding claims, wherein the lower dose of the viral vector is 5e13 or less vector genomes/kg.

42. The method or composition of any one of the preceding claims, wherein the lower dose of the viral vector is 2.5e13 or less vector genomes/kg.

43. The method or composition of any one of the preceding claims, wherein the lower dose of the viral vector is between 1-2.5e13 vector genomes/kg.

44. The method or composition of any one of the preceding claims, wherein the lower dose of the viral vector is between 1e13 or less vector genomes/kg.