Prevention or mitigation of adverse reactions associated with recombinant viral vectors

Abstract

The present invention relates to the prevention or mitigation of adverse reactions associated with gene therapy, such as the formation of anti-drug antibodies. In particular, the present invention relates to the use of tyrosine kinase inhibitors, such as dasatinib, to prevent or ameliorate such side effects.

Description

Technical Field

[0001] The present invention relates to preventing or reducing adverse reactions associated with gene therapy, such as the formation of anti-drug antibodies. In particular, the present invention relates to using tyrosine kinase inhibitors such as dasatinib to prevent or reduce such adverse reactions. Background Art

[0002] Recombinant adeno-associated virus (rAAV) or AAV vector is a viral vector used for in vivo gene therapy to deliver therapeutic transgenes into target cells. rAAV-mediated gene therapy has broad prospects for a large class of genetic diseases. Recombinant AAV capsids are usually derived from wild-type AAV, which naturally infects specific cell types according to their serotype. Recombinant AAV-mediated transgene delivery allows long-term expression of therapeutic proteins (Nathwani, AC et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. New England Journal of Medicine 371: 1994–2004 (2014)). However, rAAV gene therapy can induce immune responses to viral capsids and, in some cases, to transgenic products (Ronzitti, G. et al. Human Immune responses to Adeno-Associated Virus (AAV) vectors. Frontiers in Immunology 11: 670 (2020); Shirley, JL et al. Immune responses to viral gene therapy vectors. Molecular Therapy 28: 709-722 (2020)). Innate and adaptive immune responses may lead to cytokine release, complement activation, and cytotoxic T cell responses, but most often lead to the formation of antibodies against AAV capsids. This humoral response is characterized by the secretion of anti-AAV IgM and IgG. Most of the time, these antibodies are neutralizing antibodies that prevent any further rAAV treatment. In addition, they may cause some toxicity observed in clinical trials by mediating complement activation. For these reasons, strategies to reduce antibody formation against rAAV are highly desirable (Verdera HC et al., AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Molecular Therapy (2020)).

[0003] The tyrosine kinase inhibitor dasatinib is considered a potent compound that terminates cytokine release and T cell activation in mice treated with T cell bispecific antibodies (Leclercq et al., Journal for ImmunoTherapy of Cancer, 2021; 9(7)). Summary of the invention

[0004] The present inventors have discovered that tyrosine kinase inhibitors, in particular dasatinib, can be used to prevent the formation of anti-drug antibodies (ADA) induced by recombinant viral vector-based gene therapy.

[0005] Using an in vivo model of recombinant viral vector-based gene therapy, specifically AAV-based delivery of human proteins (hSEAP, hFactorIX) in mice, the inventors evaluated the effect of dasatinib on the formation of (undesirable) anti-drug antibodies (ADA) associated with the administration of recombinant viral vectors. Mice were intravenously administered a first rAAV8 encoding hSEAP and subsequently re-administered a second rAAV8 encoding hFactorIX. Blood samples were collected and analyzed for the presence of ADA and transgene expression. The inventors showed that dasatinib can efficiently reduce ADA formation after in vivo recombinant AAV (rAAV) administration. In addition, the inventors showed that dasatinib allows re-administration of rAAV of the same serotype. These effects can be obtained at dasatinib concentrations that are clinically relevant doses. The inventors propose that co-administration of dasatinib with recombinant viral vectors prevents the formation of ADA against the recombinant viral vector. The present invention is widely applicable to the enhancement of gene therapy treatments. For example, overcoming ADA against AAV capsids may enable repeat dosing of patients who were previously administered an AAV gene therapy product where efficacious levels had not been reached or were lost due to timing or other confounding issues.

[0006] Accordingly, in a first aspect, the present invention provides a recombinant viral vector comprising a heterologous polynucleotide for use in the treatment of a disease in an individual, wherein the treatment comprises

[0007] (a) administering the recombinant viral vector to the individual, and

[0008] (b) administering a tyrosine kinase inhibitor (TKI) to the individual for preventing or reducing the formation of anti-drug antibodies (ADA) associated with the administration of the recombinant viral vector.

[0009] The present invention further provides the use of a recombinant viral vector comprising a heterologous polynucleotide in the manufacture of a medicament for treating a disease in an individual, wherein the treatment comprises

[0010] (a) administering the recombinant viral vector to the individual, and

[0011] (b) administering a tyrosine kinase inhibitor (TKI) to the individual for preventing or reducing the formation of anti-drug antibodies (ADA) associated with the administration of the recombinant viral vector.

[0012] The present invention also provides a method for treating a disease in an individual, wherein the method comprises

[0013] (a) administering to the individual a recombinant viral vector comprising a heterologous polynucleotide, and

[0014] (b) administering a tyrosine kinase inhibitor (TKI) to the individual for preventing or reducing the formation of anti-drug antibodies (ADA) associated with the administration of the recombinant viral vector.

[0015] In another aspect, the present invention provides a tyrosine kinase inhibitor (TKI) for use in preventing or reducing the formation of anti-drug antibodies (ADA) associated with administration of a recombinant viral vector comprising a heterologous polynucleotide to a subject.

[0016] The present invention further provides the use of a tyrosine kinase inhibitor (TKI) in the manufacture of a medicament for preventing or reducing the formation of anti-drug antibodies (ADA) associated with the administration of a recombinant viral vector comprising a heterologous polynucleotide to an individual.

[0017] The present invention also provides a method for preventing or reducing the formation of anti-drug antibodies (ADA) associated with administering a recombinant viral vector comprising a heterologous polynucleotide to an individual, the method comprising administering a tyrosine kinase inhibitor (TKI) to the individual.

[0018] Unless otherwise defined herein, the terms used herein are generally as used in the art.

[0019] In some aspects, the TKI is a Lck and / or Src kinase inhibitor. In more specific aspects, the TKI is dasatinib.

[0020] "Dasatinib" is a tyrosine kinase inhibitor (TKI). It is sold under the brand name (among others) for the treatment of certain cases of chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Its CAS number, IUPAC name and chemical structure are shown below.

[0021] CAS No.: 302962-49-8

[0022] IUPAC name: N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate

[0023] Chemical Structure:

[0024]

[0025] In some aspects, (administration of) a TKI results in inhibition of the formation of anti-drug antibodies (ADA) that bind to the recombinant viral vector.

[0026] "Anti-drug antibodies" or "ADA" refer to antibodies that bind to a therapeutic agent (e.g., to AAV capsid proteins) and can affect the serum concentration and function of the therapeutic agent in an individual. The presence of ADA can increase the clearance of the therapeutic agent by forming immune complexes between the therapeutic agent and the antibody (neutralizing, non-neutralizing, or both), thereby shortening the half-life of the therapeutic agent. In addition, the activity and effectiveness of the therapeutic agent (e.g., the ability to transduce target cells) may be reduced by the binding of antibodies to the therapeutic agent (particularly in the case of neutralizing ADA). ADA may also be associated with allergic or hypersensitivity reactions and other adverse events.

[0027] "Formation of anti-drug antibodies" or "formation of ADA" refers to an immune response, particularly a humoral response, triggered by the administration of a therapeutic agent (e.g., an AAV-based recombinant viral vector) in the body of an individual. Such a response may include one or more cellular responses of T cells (particularly CD4+ T cells), such as proliferation, differentiation, cytokine secretion, and / or expression of activation markers. In addition, such a response may include activation of B cells, formation of plasma cells, formation of memory B cells, and / or secretion of antibodies (such as ADA).

[0028] In some aspects, (administration of) a TKI results in inhibition of T cell activation (induced by the recombinant viral vector).

[0029] As used herein, "activation of T cells" or "T cell activation" refers to one or more cellular responses of T lymphocytes (particularly CD4+ or CD8+ T cells) selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein. In particular aspects, T cell activation is determined by measuring the expression of CD25 and / or CD69 on T cells, for example, by flow cytometry.

[0030] In some aspects, (administration of) a TKI results in inhibition of the proliferation of T cells (induced by the recombinant viral vector).

[0031] In some aspects, (administration of) a TKI results in inhibition of the cytotoxic activity of T cells (eg, against cells expressing a polypeptide or peptide encoded by a heterologous polynucleotide introduced into the cell by a recombinant viral vector).

[0032] The "cytotoxic activity" of T cells refers to the induction of cell lysis (i.e., killing) by T lymphocytes, particularly CD8+ T cells. Cytotoxic activity generally involves degranulation of T lymphocytes, which is associated with the release of cytotoxic effector molecules such as granzyme B and / or perforin by T lymphocytes.

[0033] In some aspects, (administration of) a TKI results in inhibition of T cell receptor signaling in T cells (induced by the recombinant viral vector).

[0034] "T cell receptor signaling" refers to the activity of the downstream signaling pathway of the T cell receptor (TCR) in T lymphocytes following TCR engagement, involving signaling molecules, including tyrosine kinases, such as Lck kinase.

[0035] In some aspects, (administration of) a TKI results in inhibition of (activation of) B cells induced by the recombinant viral vector.

[0036] "Activation of B cells" refers to one or more cellular responses of B lymphocytes (particularly immature or memory B cells), selected from: proliferation, differentiation (particularly differentiation into antibody secreting effector cells, such as plasmablasts or plasma cells), antibody production, cytokine secretion, and expression of activation and / or differentiation markers. Suitable assays for measuring B cell activation are known in the art and described herein. In particular aspects, B cell activation is determined by measuring the expression of CD69 on B cells, for example, by flow cytometry.

[0037] In some aspects, TKI (administration) causes inhibition of cytokine secretion (induced by recombinant viral vector) by immune cells. In some aspects, the cytokine is one or more cytokines selected from the group consisting of the following items: IL-2, TNF-α, IFN-γ, IL-6 and IL-1β. In some aspects, the immune cell is a bone marrow cell, a CD8+T cell or a CD4+ cell.

[0038] In some aspects, the inhibition is reversible (i.e., the inhibition can be relieved so that the level of the inhibited parameter is restored to a level close to that before the inhibition). In some aspects, the inhibition is reversed after the TKI is not administered (to the individual) within a given time period (i.e., after the administration of the TKI is stopped). In some aspects, the time period is about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours.

[0039] The inhibition may be partial or complete. In some aspects, the inhibition is clinically significant and / or statistically significant.

[0040] In some aspects, TKI (administration) makes the serum level of one or more cytokines in individuality reduce.In some aspects, the one or more cytokines are selected from the group consisting of the following items: IL-2, TNF-α, IFN-γ, IL-6 and IL-1β.In some aspects, after TKI is not applied (to individual) in a given amount of time, the reduction continues.In some aspects, the amount of time is about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or 96 hours.The reduction of the serum level is particularly compared with the serum level in the individual (including the same individual) not applying TKI (i.e. in this case, compared with when TKI is not applied / before applying TKI, serum level is reduced). The reduction in serum levels is particularly compared to serum levels in individuals (including the same individual) to whom the recombinant viral vector is administered (particularly for the first time) but not to whom the TKI is administered (i.e., in this case, serum levels are reduced compared to when the recombinant viral vector is administered / after the recombinant viral vector is administered but not to whom the TKI is administered / before the TKI is administered). In the absence of the reduction, serum levels and / or cytokine secretion may be particularly increased / increased relative to (administration of) the recombinant viral vector. In some aspects, the reduction is clinically and / or statistically significant.

[0041] In some aspects, the administration of TKI is after the increase (clinical) manifestation of the serum level of one of the multiple cytokines. In some aspects, the one or more cytokines are selected from the group consisting of the following items: IL-2, TNF-α, IFN-γ, IL-6 and IL-1β. The administration can be, for example, about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or 96 hours after the increase in the serum level of one of the multiple cytokines (i.e., clinical symptoms occur, such as fever). In some aspects, the administration of TKI is reflected in the increase (clinical) manifestation of the serum level of one of the multiple cytokines (in an individual).

[0042] In some aspects, the administration of TKI is carried out before the administration of the recombinant viral vector. In some aspects, the administration of TKI is carried out simultaneously with the administration of the recombinant viral vector. In some aspects, the administration of TKI is carried out after the administration of the recombinant viral vector. In the case where the administration of TKI is carried out before the administration of the recombinant viral vector or after the administration of the recombinant viral vector, such administration of TKI can be, for example, carried out within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours or 24 hours before or after the administration of the recombinant viral vector. The administration of TKI can be intermittent administration or continuous administration. In some aspects, the administration of TKI is oral administration.

[0043] In some aspects, the dosage of TKI is enough to suppress the adverse reaction of recombinant viral vector. "Adverse reaction", sometimes also referred to as "side effect" or "adverse event" (especially in clinical studies), is the harmful and undesirable effect caused by drugs in the treatment of individuals (especially using recombinant viral vectors in this article). In some aspects, the dosage of TKI is enough to suppress the formation of ADA combined with recombinant viral vectors. In some aspects, the dosage of TKI is enough to suppress the activation of T cells (induced by recombinant viral vectors). In some aspects, the dosage of TKI is enough to suppress the cytotoxic activity of T cells (induced by recombinant viral vectors). In some aspects, the dosage of TKI is enough to suppress T cell receptor signaling in T cells (induced by recombinant viral vectors). In some aspects, the dosage of TKI is enough to suppress the cytokine secretion (induced by recombinant viral vectors) carried out by immune cells. In some aspects, the cytokine is one or more cytokines selected from the group consisting of the following items: IL-2, TNF-α, IFN-γ, IL-6 and IL-1β. In some aspects, the immune cells are bone marrow cells, CD8+T cells or CD4+ cells. The inhibition can be partial or complete. In some aspects, the inhibition has clinical significance and / or statistical significance.

[0044] In some aspects, the dosage of TKI is sufficient to suppress the production of antibodies (such as ADA) of B cells (induced by recombinant viral vectors). In some aspects, the dosage of TKI is sufficient to suppress the activation of B cells (induced by recombinant viral vectors). In some aspects, the dosage of TKI is sufficient to suppress the differentiation of B cells (induced by recombinant viral vectors). In some aspects, the dosage of TKI is sufficient to suppress the formation of plasma cells (induced by recombinant viral vectors). In some aspects, the dosage of TKI is sufficient to suppress the secretion of cytokines (induced by recombinant viral vectors) by B cells. In some aspects, the cytokine is one or more cytokines selected from the group consisting of the following items: IL-2, TNF-α, IFN-γ, IL-4, IL-6 and GM-CSF. The inhibition can be partial or complete. In some aspects, the inhibition has clinical significance and / or statistical significance.

[0045] The reduction in serum levels or cytokine secretion is particularly compared to serum levels or cytokine secretion in individuals (including the same individual) to which TKI is not administered (i.e., in this case, serum levels are reduced compared to when TKI is not administered / before TKI is administered). The reduction in serum levels or cytokine secretion is particularly compared to serum levels or cytokine secretion in individuals (including the same individual) to which TKI is administered (especially for the first time) but not TKI is administered (i.e., in this case, serum levels are reduced compared to when recombinant viral vectors are administered / after recombinant viral vectors are administered but not TKI is administered / before TKI is administered). In the absence of the reduction, serum levels and / or cytokine secretion may particularly increase / increase relative to (administering) recombinant viral vectors. In some aspects, the reduction is clinically significant and / or statistically significant. The inhibition may be partial or complete. In some aspects, the inhibition is clinically significant and / or statistically significant.

[0046] In some aspects, the TKI is administered at an effective dose.

[0047] An "effective amount" or "effective dose" of an agent, such as a TKI or a viral vector, refers to an effective amount, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive effect.

[0048] In some aspects, the dosage of the TKI is about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg or 200 mg. In some aspects, the dosage of the TKI is about 20 mg. In some aspects, the dosage of the TKI is about 70 mg. In some aspects, the dosage of the TKI is about 80 mg. In some aspects, the dosage of the TKI is about 100 mg. In some aspects, the dosage of the TKI is about 140 mg.

[0049] In some aspects, the TKI is administered at a dose of about 100 mg or less. In some aspects, the TKI is administered at a dose of about 20 mg. In some aspects, the TKI is administered at a dose of about 70 mg. In some aspects, the TKI is administered at a dose of about 80 mg. In some aspects, the TKI is administered at a dose of about 100 mg.

[0050] In some aspects, TKI is administered daily. In some aspects, TKI is administered once a day. In some aspects, TKI is administered once a day at a dosage of about 100 mg. In some aspects, the administration of TKI continues during the period in which adverse reactions persist (i.e., the administration of TKI is from the manifestation of adverse reactions to the reduction or disappearance of adverse reactions). In some aspects, the administration of TKI is stopped after the formation of ADA is prevented or reduced. In some aspects, the administration of TKI is stopped after ADA is reduced. The reduction is particularly clinically significant and / or statistically significant. In some aspects, the administration of TKI is once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times, particularly once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times during the treatment of an individual with a recombinant viral vector. In some aspects, the administration of TKI continues for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some aspects, the administration of TKI is once daily for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some aspects, the administration of TKI is associated with the first administration of the recombinant viral vector. The first administration is particularly the first administration of the recombinant viral vector in the process of treating an individual with a recombinant viral vector. In some aspects, the administration of TKI is carried out simultaneously with the first administration of the recombinant viral vector. In some aspects, the administration of TKI is carried out before the first administration of the recombinant viral vector. In some aspects, the administration of TKI is carried out after the first administration of the recombinant viral vector. In some aspects, the administration of TKI is carried out after the first administration of the recombinant viral vector and before the second administration of the recombinant viral vector. In cases where the TKI is administered before or after the (first) administration of the recombinant viral vector, the administration of this TKI can be, for example, within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours before or after the administration of the recombinant viral vector, respectively.

[0051] In some aspects, the administration of the recombinant viral vector is a single administration or repeated administration. In the process of treating an individual with a recombinant viral vector, the recombinant viral vector can be administered once or several times. In some aspects, the administration of the recombinant viral vector includes a first administration and a second administration.

[0052] In certain embodiments, (recombinant) viral vectors that can be used in the present invention include, for example, but not limited to AAV particles. In certain embodiments, viral vectors that can be used in the present invention include, for example, but not limited to retrovirus, adenovirus, helper-dependent adenovirus, hybrid adenovirus, herpes simplex virus, lentivirus, poxvirus, Epstein-Barr virus, pox virus and human cytomegalovirus vectors, including recombinant versions thereof. In a preferred embodiment, the recombinant viral vector comprises a lentiviral vector, an adenoviral vector or an adeno-associated (AAV) vector.

[0053] The term "recombinant", as a modifier of viral vectors (such as recombinant AAV (rAAV) vectors) and modifiers of sequences (such as recombinant polynucleotides and polypeptides) means that the composition has been manipulated (i.e., engineered) in a manner that does not normally occur in nature. A specific example of a recombinant AAV vector would be the insertion of a nucleic acid (heterologous polynucleotide) that is not normally present in the wild-type AAV genome into the viral genome. An example would be a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence cloned into a vector with or without the 5', 3', and / or intron regions to which the gene is normally associated in the AAV genome. Although the term "recombinant" is not always used herein to refer to AAV vectors and sequences such as polynucleotides, recombinant forms including AAV vectors, polynucleotides, etc. are expressly included, regardless of any such omissions.

[0054] For example, a "rAAV vector" is derived from the wild-type genome of AAV by using molecular methods to remove all or part of the wild-type AAV genome and replace it with a non-natural (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. In general, for rAAV vectors, one or two inverted terminal repeat (ITR) sequences of the AAV genome are retained. rAAV is different from the AAV genome because all or part of the AAV genome has been replaced with a non-natural sequence relative to the AAV genome nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Therefore, the incorporation of non-natural (heterologous) sequences defines AAV as a "recombinant" AAV vector, which can be referred to as a "rAAV vector".

[0055] The recombinant AAV vector sequence can be packaged, referred to herein as a "particle", which is used to subsequently infect (transduce) cells in vitro, in vitro or in vivo. In the case where the recombinant vector sequence is encapsidated or packaged into an AAV particle, the particle may also be referred to as "rAAV", "rAAV particle" and / or "rAAV virion". Such rAAV, rAAV particles and rAAV virions include proteins that encapsulate or package the vector genome. Specific examples include, in the case of AAV, including capsid proteins.

[0056] "Vector genome", which may be abbreviated as "vg", refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form rAAV particles. In the case where a recombinant plasmid is used to construct or prepare a recombinant AAV vector, the AAV vector genome does not include a "plasmid" portion that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called a "plasmid backbone", which is important for cloning and amplification of the plasmid (a process required for propagation and production of recombinant AAV vectors), but is not itself packaged or encapsidated into rAAV particles. Therefore, "vector genome" refers to a nucleic acid that is packaged or encapsidated by rAAV.

[0057] As used herein, the term "serotype" with respect to an AAV vector refers to a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to antibodies to another AAV. Differences in cross-reactivity are typically due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Due to homology in capsid protein sequences, antibodies against one AAV may cross-react with one or more other AAV serotypes.

[0058] Under the traditional definition, serotype refers to the target virus that has been tested for serum, which has specificity for neutralizing activity to all existing and characterized serotypes, and no antibodies to neutralize the target virus are found. With the discovery of more naturally occurring virus isolates and / or the generation of capsid mutants, there may or may not be serological differences with any of the currently existing serotypes. Therefore, in the case where a new virus (such as AAV) does not have serological differences, the new virus (such as AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not yet been performed on mutant viruses with modified capsid sequences to determine whether they have another serotype according to the traditional definition of serotype. Accordingly, for convenience and to avoid repetition, the term "serotype" refers broadly to serologically different viruses (such as AAV) and serologically no different viruses (such as AAV), which may be in a subgroup or variant of a given serotype.

[0059] rAAV viral vectors include any strain or serotype. For example, but not limited to, rAAV vector genomes or particles (capsids, such as VP1, VP2 and / or VP3) can be based on any AAV serotype, such as, for example, AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rhlO, AAV3B or AAV-2i8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or different from each other. For example, but not limited to, rAAV plasmids or vector genomes or particles (capsids) based on a serotype genome can be the same as one or more capsid proteins of the packaging vector. In addition, the rAAV plasmid or vector genome can be based on an AAV serotype genome that is different from one or more capsid proteins that package the vector genome, in which case at least one of the three capsid proteins can be a different AAV serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8, or a variant thereof. More specifically, the rAAV2 vector genome can comprise the AAV2 ITRs but comprise a capsid from a different serotype, such as, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8, or a variant thereof. Accordingly, rAAV vectors include gene / protein sequences identical to those characteristic of a particular serotype as well as "hybrid" serotypes (which may also be referred to as "pseudotypes").

[0060] In certain embodiments, the rAAV plasmid or vector genome or particle is based on a reptile or invertebrate AAV variant, such as snake and lizard parvovirus (Penzes et al., 2015, J. Gen. Virol., 96:2769-2779) or insect and shrimp parvovirus (Roekring et al., 2002, Virus Res., 87:79-87).

[0061] In certain embodiments, the recombinant plasmid or vector genome or particle is based on a Pocavirus variant. Human Pocavirus variants are described, for example, in Guido et al., 2016, World J. Gastroenterol., 22: 8684-8697.

[0062] In one embodiment, the recombinant viral vector comprises a protein to which ADA binds. In one embodiment, the recombinant lentiviral vector comprises an envelope protein to which ADA binds. In one embodiment, the recombinant AAV (rAAV) vector comprises a capsid protein to which ADA binds.

[0063] In one embodiment, the recombinant AAV (rAAV) vector comprises VP1, VP2 and / or VP3 capsid proteins having 70% or greater sequence identity to VP1, VP2 and / or VP3 capsid proteins selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8 VP1, VP2 and / or VP3 capsid proteins. In one embodiment, the recombinant AAV (rAAV) vector comprises VP1, VP2 and / or VP3 capsid proteins having 100% sequence identity with VP1, VP2 and / or VP3 capsid proteins selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8VP1, VP2 and / or VP3 capsid proteins. In certain embodiments, the AAV vector comprises or consists of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, or AAV3B, ITR.

[0064] In certain embodiments, recombinant AAV (rAAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, RhlO, Rh74 and AAV-2i8 variants (e.g., ITRs and capsid variants thereof, such as amino acid insertions, additions, substitutions and deletions), for example, as described in WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO 2015 / 013313 (International Application PCT / US2014 / 047670) and US2013 / 0059732 (U.S. Application No. 13 / 594,773).

[0065] In a preferred embodiment, the recombinant viral vector is selected from the group consisting of: AAV2, AAV8 and AAV9. In one such preferred embodiment, the recombinant AAV (rAAV) vector is selected from the group consisting of: rAAV2, rAAV8 and rAAV9. In one such preferred embodiment, the AAV vector comprises VP1, VP2 and / or VP3 capsid proteins having 70% or more sequence identity with the VP1, VP2 and / or VP3 capsid proteins selected from the group consisting of: AAV2, AAV8 and AAV8 VP1, VP2 and / or VP3 capsid proteins. In one such preferred embodiment, the AAV vector comprises VP1, VP2 and / or VP3 capsid proteins having 100% or more sequence identity with the VP1, VP2 and / or VP3 capsid proteins selected from the group consisting of: AAV2, AAV8 and AAV8 VP1, VP2 and / or VP3 capsid proteins.

[0066] 24. A recombinant viral vector, TKI, use or method according to claim 19, 21 or 22, wherein the AAV vector comprises a VP1, VP2 and / or VP3 capsid protein having 100% sequence identity with a VP1, VP2 and / or VP3 capsid protein selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8 VP1, VP2 and / or VP3 capsid protein

[0067] rAAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8 and variants, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to include one or more heterologous polynucleotide sequences (transgenes) flanked by one or more functional AAV ITR sequences. Such AAV vectors typically retain at least one functional flanking ITR sequence, such as necessary for rescue, replication and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome will include cis sequences (e.g., functional ITR sequences) required for replication and packaging.

[0068] In certain embodiments, the lentivirus used in the present invention can be human immunodeficiency-1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease virus (JDV), equine infectious anemia virus (EIAV) or caprine arthritis encephalitis virus (CAEV). Lentiviral vectors can provide efficient delivery, integration and long-term expression of heterologous polynucleotide sequences in vitro and in vivo to non-dividing cells. A variety of lentiviral vectors are known in the art, see Naldini et al. (Proc. Natl. Acad. Sci. USA, 93: 11382-11388 (1996); Science, 272: 263-267 (1996)), Zufferey et al., (Nat. Biotechnol, 15: 871-875, 1997), Dull et al., (J Virol. 1998 November; 72 (11): 8463-71, 1998), U.S. Pat. Nos. 6,013,516 and 5,994,136, any of which may be a suitable viral vector for use in the present invention.

[0069] An immune response, such as humoral immunity, can also be generated against the recombinant viral vector and / or the heterologous polynucleotide or the protein or peptide encoded by the heterologous polynucleotide encapsidated by the viral vector, thereby resulting in inhibition or reduction of viral vector cell transduction, heterologous polynucleotide expression or function, or the function or activity of the protein or peptide encoded by the heterologous polynucleotide in the subject to which the viral vector is administered.

[0070] Antibodies (such as ADA) (which may be referred to as "neutralizing" antibodies) that bind to viral vectors (such as recombinant viral vectors) used in the present invention can reduce or inhibit cell transduction of viral vectors that can be used for gene therapy. Thus, while not being bound by theory, cell transduction is reduced or inhibited, thereby reducing the introduction and subsequent expression of virally packaged heterologous polynucleotides into cells, and subsequent translation to proteins or peptides if appropriate. In addition, antibodies that bind to heterologous polynucleotides or proteins or peptides encoded by heterologous polynucleotides encapsidated by viral vectors can inhibit the expression of heterologous polynucleotides, the function or activity of heterologous polynucleotides, or the function or activity of proteins or peptides encoded by heterologous polynucleotides.

[0071] Accordingly, there may be antibodies (such as ADA) that bind to a recombinant viral vector (e.g., AAV) and / or there may be antibodies that bind to a protein or peptide encoded by a heterologous polynucleotide in a subject. In addition, there may be antibodies that bind to a heterologous polynucleotide encapsidated by a recombinant viral vector.

[0072] Antibodies that bind to a recombinant viral vector (e.g., AAV) or to a protein or peptide encoded by a heterologous polynucleotide, if induced, can be reduced or eliminated in a subject by using a tyrosine kinase inhibitor (TKI) as described herein.

[0073] In one embodiment, ADA comprises IgG, IgM, IgA, IgD and / or IgE. IgG, IgM, IgA, IgD and / or IgE antibodies combined with a recombinant viral vector (e.g., rAAV) or combined with a protein or polypeptide encoded by a heterologous polynucleotide, if induced, can be reduced or eliminated in a subject by using a tyrosine kinase inhibitor (TKI) as described herein. The reduction of circulating antibodies (e.g., reduction of antibody levels in blood, plasma or serum) can be measured by standard assays known in the art and as described herein. In one embodiment, ADA comprises IgG and / or IgG.

[0074] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA tRNA, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, micro RNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA).

[0075] Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length. When discussing nucleic acids, the sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0076] "Heterologous" polynucleotide or nucleic acid sequence refers to a polynucleotide inserted into a plasmid or vector for the purpose of vector-mediated transfer / delivery of polynucleotides to cells. Heterologous nucleic acid sequences are different from viral nucleic acids, that is, they are non-natural relative to viral nucleic acids. Once transferred / delivered to the cell, the heterologous nucleic acid sequence contained in the vector can be expressed (e.g., transcribed, and if appropriate, translated). Alternatively, the heterologous polynucleotide transferred / delivered in the cell contained in the expression vector is not required. Although the term "heterologous" is not always used to refer to nucleic acid sequences and polynucleotides in this article, even in the absence of the modifier "heterologous", it is intended to include heterologous nucleic acid sequences and polynucleotides to refer to nucleic acid sequences or polynucleotides, regardless of whether there is an omission.

[0077] "Transgenic" is used herein to refer to a nucleic acid that is intended to or has been introduced into a cell or organism. A transgenic includes any nucleic acid, such as a heterologous polynucleotide sequence or a heterologous nucleic acid encoding a protein or peptide. The terms transgenic and heterologous nucleic acid / polynucleotide sequence are used interchangeably herein.

[0078] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to an attempt to alter the natural course of the disease in the individual being treated, and may be performed for prevention or may be performed during clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, promoting or alleviating the disease state, and alleviating or improving prognosis.

[0079] In one embodiment, the subject has a lung disease (e.g., cystic fibrosis), a bleeding disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), a thalassemia, a blood disorder (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, a lysosomal storage disease (e.g., aspartylglucosaminuria, Batten disease, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2 gangliosidosis type I (Tay-Sachs disease), GM2 gangliosidosis type II (Sandhoff disease), mucolipidosis type I (salivary gland disease types I and II), type II (I cell disease), type III (pseudo-Hurler disease) and type IV, mucopolysaccharidosis (Hurler disease and variants, Hunter, Sanfilippo A, B, C, D, Morquio A and B, Maroteaux-Lamy and Sly disease), Niemann-Pick disease A / B, C1 and C2, and Schild disease types I and II), hereditary angioedema (HAE), copper or iron storage disorders (e.g., Wilson disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), diseases of solid organs (e.g., brain, liver, kidney, heart), or infectious viral (e.g., hepatitis B and C, HIV, etc.), bacterial or fungal diseases.

[0080] In one embodiment, the individual suffers from a coagulation disorder. In one embodiment, the individual suffers from hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any coagulation factor (VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor) or a combined deficiency of FV / FVIII, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency.

[0081] In one embodiment, the individual suffers from anemia; bleeding associated with trauma, pentasaccharide, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic drugs (i.e., FXa inhibitors), or a platelet disorder such as pediatric giant platelet syndrome, thrombasthenia, storage pool deficiency.

[0082] In one embodiment, the individual suffers from a disease that affects or originates in the central nervous system (CNS). In one embodiment, the disease is a neurodegenerative disease. In one embodiment, the CNS or neurodegenerative disease is Alzheimer's disease, Huntington's disease, AFS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, polyglutamine repeat disease, or Parkinson's disease. In one embodiment, the CNS or neurodegenerative disease is a polyglutamine repeat disease. In one embodiment, the polyglutamine repeat disease is spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0083] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of: GAA (acid alpha-glucosidase) for treating Pompe disease; ATP7B (copper-transporting ATPase 2) for treating Wilson disease; alpha-galactosidase for treating Fabry disease; ASS1 (arginine succinate synthase) for treating citrullinemia type 1; beta-glucocerebrosidase for treating Gaucher disease type 1; beta-hexosaminidase A for treating Tay-Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor, also known as C1 inhibitor deficiency type I and type II) for treating hereditary angioedema (HAE); and glucose-6-phosphatase for treating glycogen storage disease type I (GSDI).

[0084] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiogenin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a The proteins involved in the production of these proteins are: platelet-derived growth factor (PDGF), insulin-like growth factor I and II (IGF-I and IGF-II), transforming growth factor beta (TGF-β), activins, inhibins, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, axon guidance factor-1 and axon guidance factor-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0085] In certain embodiments, the heterologous polynucleotide encodes acid alpha-glucosidase (GAA). Administration of a recombinant viral vector comprising a heterologous polynucleotide encoding GAA to a subject with Pompe disease or another glycogen storage disease may result in expression of the GAA protein. Expression of the GAA protein in the patient can be used to suppress, inhibit or reduce the accumulation of glycogen, prevent the accumulation of glycogen, or degrade glycogen, which in turn can reduce or reduce one or more adverse reactions of Pompe disease or another glycogen storage disease.

[0086] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of thrombopoietin (TPO), interleukins (IL-1 to IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor a and b, interferon alpha, beta and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

[0087] In certain embodiments, the heterologous polynucleotide encodes CFTR (cystic fibrosis transmembrane regulator), blood coagulation (coagulation) factors (factor XIII, factor IX, factor VIII, factor X, factor VII, factor Vila, protein C, etc.) gain-of-function blood coagulation factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, etc. factors, neurotrophic factors-3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor alpha and beta, cytokines, interferon-alpha, interferon-beta, interferon-g, interleukin-2, interleukin-4, interleukin-12, granulocyte macrophage colony stimulating factor, lymphotoxin, suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, Hippel-Lindau syndrome (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (choroiderem), LCA 5 (LCA-Lebercilin), ornithine ketoacid transaminase (ring atrophy), retinoschisis protein 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (RP: AR form of retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (achromatopsia), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin A, GM2-AP, NPC1, VPC2, saposhin, one or more zinc finger nucleases for genome editing, or one or more donor sequences for use as a repair template for genome editing.

[0088] In certain embodiments, the heterologous polynucleotide encodes erythropoietin (EPO) for treating anemia; interferon-α, interferon-β and interferon-γ for treating various immune disorders, viral infections and cancers; interleukins (IL), including any one of IL-1 to IL-36, and corresponding receptors, for treating various inflammatory diseases or immune deficiencies; chemokines, including chemokine (CXC motif) ligand 5 (CXCL5), for treating immune disorders; granulocyte-macrophage colony stimulating factor (G-CSF), for treating immune disorders, such as Crohn's disease; granulocyte-macrophage colony stimulating factor (G-CSF), for treating immune disorders, such as Crohn's disease; GM-CSF, used to treat a variety of human inflammatory diseases; macrophage colony stimulating factor (M-CSF), used to treat a variety of human inflammatory diseases; keratinocyte growth factor (KGF), used to treat epithelial tissue damage; chemokines, such as monocyte chemoattractant protein 1 (MCP-1), used to treat recurrent miscarriage, HIV-related complications and insulin resistance; tumor necrosis factor (TNF) and receptors, used to treat various immune disorders; alpha-1-antitrypsin, used to treat emphysema or chronic obstructive pulmonary disease (COPD); alpha-L-iduronidase, used to treat mucopolysaccharidosis I (MPS I); ornithine transcarbamylase (OTC), used to treat OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine deaminase (PAL), used to treat phenylketonuria (PKU); lipoprotein lipase, used to treat lipoprotein lipase deficiency; apolipoprotein, used to treat apolipoprotein (Apo) AI deficiency; low-density lipoprotein receptor (LDL-R), used to treat familial hypercholesterolemia (FH); albumin, used to treat hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate synthetase; argininosuccinate Lyase; arginase; fumaryl acetoacetate hydrolase; porphobilinogen deaminase; cystathionine beta-synthase, used to treat homocystinuria; branched-chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl-CoA carboxylase; methylmalonyl-CoA mutase; glutaryl-CoA dehydrogenase; insulin; pyruvate carboxylase; liver phosphorylase; phosphorylase kinase; glycine decarboxylase; H-protein; T-protein; cystic fibrosis transmembrane regulator (CFTR); ATP-binding cassette, subfamily A (ABC1), member 4 (ABCA4), used to treat Stargardt disease; or muscular dystrophy protein.

[0089] The terms "polypeptide", "protein" and "peptide" are used interchangeably herein. "Polypeptides", "proteins" and "peptides" encoded by "polynucleotide sequences" include full-length native sequences, such as naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants, as long as the subsequences, modified forms or variants retain a certain degree of functionality of the native full-length protein. In the present invention, such polypeptides, proteins and peptides encoded by polynucleotide sequences can be, but are not required to be, identical to endogenous proteins that are defective, under-expressed or missing in the mammal being treated.

[0090] In certain embodiments, the heterologous polynucleotide encodes an inhibitory nucleic acid selected from the group consisting of: siRNA, antisense molecules, miRNA, RNAi, ribozymes, and shRNA.

[0091] In certain embodiments, the inhibitory nucleic acid binds to a gene, a transcript of a gene, or a transcript of a gene associated with a multinucleotide repeat disease selected from the group consisting of: huntingtin (HTT) gene; a gene associated with dentatorubropallidoluysian atrophy (atrophin 1, ATN1); androgen receptor on chromosome X in spinobulbar muscular atrophy; human ataxin 1, 2, 3, and 7; Cav2.1 P / Q voltage-dependent calcium channel (CACNA1A); TATA binding protein; ataxin 8 opposite chain (ATXN8OS); serine / threonine protein phosphatase 2A 55 kDa regulatory subunit B beta isoform (1, 2, 3, 6, 7, 8, 12) in spinocerebellar ataxia 17); FMR1 (fragile X mental retardation 1) in fragile X syndrome; FMR1 (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome; FMR1 (fragile X mental retardation 2) or AF4 / FMR2 family member 2 in fragile XE mental retardation; myotonic protein kinase (MT-PK) in myotonic dystrophy; tataxin in Friedreich's ataxia; mutants of superoxide dismutase 1 (SOD1) in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator of transcription gene in HIV infection; HIV TAR, HIV in HIV infection TAR, human immunodeficiency virus transactivator response element gene; CC chemokine receptor (CCR5) in HIV infection; RSV nucleocapsid protein in Rous sarcoma virus (RSV) infection; liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function in renal transplantation or acute renal failure in renal injury; protein kinase N3 (PKN3) in late recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta-9 type (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-8 type (PSMB 8), metastatic melanoma; MECL1, also known as proteasome subunit beta-10 type (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; spindle kinesin in solid tumors, apoptosis inhibitor B cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors;Polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2-adrenergic receptor, glaucoma; RTP801 / Redd 1, also known as DNA damage-inducible transcript 4 protein, in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in nonarteritic ischemic optic neuropathy; keratin 6A in pachyonychia congenita N17K mutant protein; influenza A virus genome / gene sequence in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence in SARS infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola filovirus genome / gene sequence in Ebola virus infection; hepatitis B and C virus genome / gene sequence in hepatitis B and C infection; herpes simplex virus (HSV) genome / gene sequence in HSV infection, Coxsackie B3 virus genome / gene sequence in Coxsackie B3 virus infection; silencing of pathogenic alleles of genes (such as tension protein A (TORI A) in primary muscle tone deficiency, specific pan-class I and HLA alleles in transplantation) (allele-specific silencing); and mutant rhodopsin gene (RHO) in orthosomal dominant hereditary pigmentary retinitis (adRP). ;

[0092] In one embodiment, the protein encoded by the heterologous polynucleotide comprises a gene editing nuclease. In one embodiment, the gene editing nuclease comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). In one embodiment, the gene editing nuclease comprises a functional type II CRISPR-Cas9.

[0093] For use in the present invention, the recombinant viral vector will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the recombinant viral vector, the method of administration, the timing of administration, and other factors known to practitioners.

[0094] The recombinant viral vector of an effective amount can be administered to prevent or treat the disease. The appropriate route of administration and dosage of the recombinant viral vector can be determined based on the type of disease to be treated, the type of the recombinant viral vector, the severity and course of the disease, the clinical condition of the individual, the clinical history of the individual and the response to treatment and the judgment of the attending physician. Administration can be carried out by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations, push administrations, and pulse infusions at various time points.

[0095] The recombinant viral vector may be administered at any suitable dose. Generally, the dose range will be at least 1 x 10 8 or more, e.g. 1x10 9 , 1x10 10 , 1x1011, 1x10 12 , 1x10 13 or 1x10 14 or more vector genomes (vg / kg) to achieve therapeutic effect. In mice, the range is 1x10 10 Up to 1x10 11 vg / kg and range of 1x10 in dogs 12 Up to 1x10 13 More specifically, a dose of about 1 x 10 11 vg / kg to about 5x10 14 vg / kg (inclusive), or about 5x10 11 vg / kg to about 1x10 14 vg / kg (inclusive), or about 5x10 11 vg / kg to about 5x10 13 vg / kg (inclusive), or about 5x10 11 vg / kg to about 1x10 13 vg / kg (inclusive), or about 5x10 11 vg / kg to about 5x10 12 (including the end value), or about 5x10 11 About 1x10 12 vg / kg (inclusive of the end value). The dosage can be, for example, about 5x10 14 vg / kg, or less than about 5x10 14 vg / kg, such as a dose of about 2x10 11 About 2x10 14 vg / kg (including end values), in particular, for example, about 2x10 12 vg / kg, about 6x1012 vg / kg or about 2x10 13 vg / kg.

[0096] Dosage can vary and depends on the type, onset, progression, severity, frequency, duration or probability of the disease for which treatment is directed, the desired clinical endpoint, previous or simultaneous treatment, the overall health, age, sex, race or immune competence of the subject, and other factors that will be understood by those skilled in the art. The dosage, number, frequency or duration can be increased or decreased in proportion, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the state of the subject. Those skilled in the art will understand the factors that may affect the dosage and timing required to provide an amount sufficient to provide a therapeutic or preventive benefit.

[0097] The dosage to achieve a therapeutic effect (e.g., a dosage in units of vector genomes / kg body weight (vg / kg)) will vary based on a variety of factors, including, but not limited to: the route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease being treated, any host immune response to the recombinant viral vector, the host immune response to the heterologous polynucleotide or expression product (protein or peptide or transcribed nucleic acid), and the stability of the expressed protein or peptide or transcribed nucleic acid. A skilled artisan can determine the range of recombinant viral vector genome dosages for treating patients with a particular disease or condition based on the foregoing factors and other factors.

[0098] An "effective amount" or "sufficient amount" refers to a single dose or multiple doses, alone or in combination with one or more other compositions, treatments, regimens or treatment plans, that provides a detectable response of any duration (long-term or short-term), an expected or desired result in a subject, or any measurable or detectable degree or benefit to a subject for any duration (e.g., for minutes, hours, days, months, years, or a cure). An "effective amount" or "sufficient amount" dose for treatment (e.g., to enhance or provide a therapeutic benefit or improvement) is generally effective to provide a response to one, more or all adverse symptoms, consequences or complications of a disease, such as one or more adverse symptoms, disorders, conditions, pathologies or complications caused by or associated with the disease to a measurable degree, although reducing, diminishing, inhibiting, suppressing, limiting or controlling the progression or worsening of the disease is a satisfactory result.

[0099] The recombinant viral vector and TKI can be administered by any suitable route, and can be administered by the same route of administration or by different routes of administration. In some aspects, administration of the recombinant viral vector is parenteral administration, particularly intravenous administration.

[0100] In some aspects, the administration of the recombinant viral vector is the first administration of the recombinant viral vector to the individual, particularly the first administration of the recombinant viral vector in a course of treating the individual with the recombinant viral vector.

[0101] An effective amount or sufficient amount may, but need not, be provided in a single administration, may require multiple administrations, and may, but need not, be administered alone or in combination with another composition (e.g., medicament), treatment, regimen, or treatment plan. For example, the amount may be increased proportionally as indicated by the needs of the subject, the type, state, and severity of the disease being treated, or the side effects of the treatment (if any). In addition, if administered in a single or multiple doses without a second composition (e.g., another drug or medicament), treatment, regimen, or treatment plan, an effective amount or sufficient amount need not be effective or sufficient, because additional doses, amounts, or durations above and beyond such doses, or additional compositions (e.g., drugs or medicaments), treatments, regimens, or treatment plans may be included in order to be considered effective or sufficient in a given subject. An amount considered effective also includes an amount that results in a reduction in the use of another treatment, treatment plan or regimen, such as the administration of recombinant GAA to treat a lysosomal storage disease (e.g., Pompe disease), or the administration of a recombinant coagulation factor protein (e.g., FVIII or FIX) to treat a coagulation disorder (e.g., hemophilia A (HemA) or hemophilia B (HemB)).

[0102] For Pompe disease, an effective amount will be, for example, an amount of GAA that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal, reduces lysosomal changes in tissues of the subject's body, or improves muscle tone and / or muscle strength and / or respiratory function in the subject. For example, an effective amount can be determined by determining the kinetics of GAA uptake from plasma by myoblasts. Myoblasts with a GAA uptake rate (potassium uptake) of about 141-147 nM may be effective (see, e.g., Maga et al., J. Biol. Chem. 2012). In animal models, GAA activity levels in plasma greater than about 1,000 nmol / hr / mL, e.g., about 1,000 to about 2,000 nmol / hr / mL have been observed to have a therapeutic effect.

[0103] For HemA and HemB, in general, it is believed that in order to achieve a therapeutic effect, a coagulation factor concentration greater than 1% of the coagulation factor concentration found in normal individuals is required to change the severe disease phenotype to a moderate disease phenotype. The severe phenotype is characterized by joint damage and life-threatening bleeding. In order to convert the moderate disease phenotype to a mild disease phenotype, a coagulation factor concentration greater than 5% of the normal coagulation factor concentration is believed to be required.

[0104] The level of FVIII and FIX in normal humans is about 150 to 200 ng / mL plasma, but may be lower (e.g., in the range of about 100 to 150 ng / mL) or higher (e.g., in the range of about 200 to 300 ng / mL) and still considered normal due to functional coagulation as determined, for example, by the activated partial thromboplastin time (aPTT) one-stage coagulation assay. Thus, a therapeutic effect can be achieved such that the total amount of FVIII or FIX in a subject / human is greater than 1%, e.g., 1% of 100 to 300 ng / mL of the FVIII or FIX present in a normal subject / human.

[0105] The composition can be administered to a subject as a combined composition or separately, such as simultaneously or consecutively or sequentially with the delivery or administration of a recombinant viral vector comprising a heterologous polynucleotide (before or after). The invention provides combinations in which the methods or uses of the invention are combined with any compound, agent, medicament, treatment plan, treatment regimen, process, therapy, or composition described herein or known to those skilled in the art. The compound, agent, medicament, treatment plan, treatment regimen, process, therapy, or composition can be administered or performed before, after, or substantially simultaneously with the administration of a recombinant viral vector comprising a heterologous polynucleotide to a subject.

[0106] An effective amount or sufficient amount need not be effective for every subject treated, nor need it be effective for a majority of subjects treated in a given group or population. An effective amount or sufficient amount refers to effectiveness or adequacy for a specific subject rather than a group or general population. As is typical for such methods, some subjects will exhibit a higher response, or a lower or zero response to a given treatment method or use.

[0107] The term "enhance" refers to a detectable or measurable improvement in a subject's disease or its symptoms or potential cellular responses. A detectable or measurable improvement includes a subjective or objective reduction, reduction, inhibition, suppression, restriction or control of the occurrence, frequency, severity, progression or duration of a disease or a complication caused by or associated with the disease, or an improvement in the symptoms or underlying causes or results of the disease, or a turnaround in the disease. For Pompeii, an effective amount will be, for example, an amount that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal, improves muscle tone and / or muscle strength and / or respiratory function. For HemA or HemB, an effective amount will be, for example, an amount that reduces the frequency or severity of acute bleeding episodes in a subject, or reduces, for example, an amount of clotting time as measured by a coagulation assay.

[0108] Accordingly, the pharmaceutical compositions of the present invention include compositions wherein the active ingredients are contained in effective amounts to achieve the intended therapeutic purpose. It is well within the capabilities of a skilled medical practitioner to determine a therapeutically effective dose using techniques and guidance known in the art and using the teachings provided herein.

[0109] The therapeutic dose will depend, among other factors, on the age and general condition of the subject, the severity of the abnormal phenotype, and the strength of the control sequences regulating the expression level. Thus, the therapeutically effective amount in humans will fall within a relatively wide range that can be determined by the medical practitioner based on the individual patient's response to vector-based therapy. Such doses may be alone or in combination with an immunosuppressant or drug.

[0110] Compositions such as pharmaceutical compositions can be delivered to a subject to allow for expression of the transgene and optionally production of the encoded protein. In certain embodiments, the pharmaceutical composition comprises sufficient genetic material to enable the subject to produce a therapeutically effective amount of a coagulation factor to improve hemostasis in the subject. In certain embodiments, the pharmaceutical composition comprises sufficient heterologous polynucleotides to enable the subject to produce a therapeutically effective amount of GAA.

[0111] In certain embodiments, the therapeutic effect in the subject is maintained for a period of time, such as 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, or 30-50 days or longer, such as 50-75, 75-100, 100-150, 150-200 days or longer. Accordingly, in certain embodiments, the recombinant viral vector provides a therapeutic effect.

[0112] "Individuals" or "subjects" herein are mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human. In some aspects, the individual suffers from a disease, particularly a disease that is treatable or to be treated by a recombinant viral vector.

[0113] In some aspects, the serum level of one or more cytokines of an individual increases. In some aspects, the increase in the serum level is relevant to the application of a recombinant viral vector to an individual. The increase in the serum level is particularly compared to the serum level in the serum level of a healthy individual and / or the serum level in an individual (including the same individual) to which a recombinant viral vector is not applied (i.e., in this case, compared to the serum level to which a recombinant viral vector is not applied, the serum level increases). In some aspects, the one or more cytokines are selected from the group consisting of the following items: IL-2, TNF-α, IFN-γ, IL-6, and IL-1β.

[0114] The cytokine according to any aspect of the present invention is preferably a proinflammatory cytokine, in particular one or more cytokines selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6 and IL-1β. In some aspects, the cytokine is IL-2. In some aspects, the cytokine is TNF-α. In some aspects, the cytokine is IFN-γ. In some aspects, the cytokine is IL-6. In some aspects, the cytokine is IL-1β.

[0115] In some aspects, treatment with a recombinant viral vector or administration of a recombinant viral vector can result in a reaction in an individual. In some aspects, the reaction can be a complete reaction. In some aspects, the reaction can be a sustained reaction after treatment stops. In some aspects, the reaction can be a complete reaction that lasts after treatment stops. In other aspects, the reaction can be a partial reaction. In some aspects, the reaction can be a partial reaction that lasts after treatment stops. In some aspects, compared with the treatment of a recombinant viral vector alone or the administration of a recombinant viral vector (i.e., without TKI), the treatment of a recombinant viral vector or the administration of a recombinant viral vector and TKI can improve reaction. In some aspects, compared with the corresponding patient population treated with a recombinant viral vector alone (i.e., without TKI), the treatment or administration of a recombinant viral vector and TKI can increase the response rate in the patient population.

[0116] In one aspect, the individual is at risk of producing ADA that binds to the recombinant viral vector. In one embodiment, before and / or after the administration of the TKI, the individual does not have ADA that binds to the recombinant viral vector. The method of measuring ADA before and / or after the administration of the recombinant viral vector is known in the art and is also described herein.

[0117] In one embodiment, the ADA combined with the recombinant viral vector is reduced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, optionally compared to the natural history data of the relevant control group of the tyrosine kinase inhibitor (TKI) not being administered. In one embodiment, the reduction of ADA in the serum of an individual is measured. The reduction of the serum level of ADA is particularly compared to the serum level in individuals (including the same individual) to which TKI is not administered (i.e., in this case, compared to the serum level before / when TKI is not administered, the serum level is reduced). The reduction of the serum level is particularly compared to the serum level or cytokine secretion in individuals (including the same individual) to which TKI is administered (especially for the first time) but not administered (i.e., in this case, compared to the administration of the recombinant viral vector / administration of the recombinant viral vector after but not administered TKI / administration of the TKI before, the serum level is reduced). In the absence of the reduction, the serum level (of ADA) may be increased / increased relative to (administration of) the recombinant viral vector. In some aspects, the reduction has clinical significance and / or statistical significance. The reduction can be partial or complete. In some aspects, the reduction is clinically significant and / or statistically significant.

[0118] In one embodiment, the individual is at risk for producing ADA to a polypeptide encoded by the heterologous polynucleotide.ADA to a heterologous polypeptide can reduce the efficacy of a treatment, for example, by reducing the number of cells expressing the heterologous polypeptide (ie, the transgene).

[0119] In one embodiment, transgenic expression increases when the viral vector is re-administered, particularly compared to transgenic expression before the viral vector is re-administered. In one embodiment, wherein compared to transgenic expression before the viral vector is re-administered, transgenic expression increases by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In one embodiment, transgenic expression is maintained when the viral vector is re-administered, particularly compared to transgenic expression before the viral vector is re-administered. In one embodiment, the increase in transgenic expression is measured in the serum of an individual and / or in a (target) tissue. The increase in transgenic expression is particularly compared to transgenic expression in individuals (including the same individual) to which TKI is not administered (i.e., in this case, compared to transgenic expression before / when TKI is not administered, transgenic expression is reduced). The increase in transgenic expression is particularly compared to transgenic expression in individuals (including the same individual) to which a recombinant viral vector is administered (especially for the first time) but not administered with a TKI (i.e., in this case, compared to administration of a recombinant viral vector / administration of a recombinant viral vector but not administered with a TKI / before administration of a TKI, transgenic expression increases). In the absence of the increase, transgenic expression may be reduced / decreased relative to (administration of) the recombinant viral vector. In some aspects, the increase is clinically and / or statistically significant. The increase may be partial or complete. In some aspects, the increase is clinically and / or statistically significant.

[0120] sequence

[0121]

[0122]

[0123] BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 .rAAV8 re-administration and dasatinib treatment in mice. C57Bl / 6 mice were intravenously administered rAAV8 encoding hSEAP (human secretory alkaline phosphatase) at a dose of 1E12vg / kg on study day 1. On study day 22, they received a second rAAV8 encoding hFIX (human factor IX) at a dose of 5E13vg / kg. Group 1 (unimmunized mice, n=9) did not receive the first rAAV8 injection. Group 2 (immunized mice, n=10) received two rAAV8 injections. Group 3 mice (n=10) received two rAAV8 injections and were treated with 50mg / kg dasatinib by gavage twice a day for 7 days starting on the day of the first rAAV8 administration. Blood samples were collected one day before the first rAAV8 treatment and every 7 days thereafter.

[0125] Figure 2. Effect of dasatinib treatment on anti-AAV8 IgM titers. Serum was isolated from blood collected on study days 0, 8, 15, 21, 29, and 36 ( Figure 1 ), and anti-AAV8 IgM was measured by ELISA performed on immobilized empty AAV8 capsids. IgM antibody titers were determined by ELISA OD values ​​of serial dilutions (1:10 and 7-step serial dilution 1:3). To determine the average background content, readings were pooled from all mouse groups on day 0. The positive threshold is represented by the dotted line. The kinetics of anti-AAV8 IgM antibody titers are shown for each mouse group ( Figure 2A-2C . Open circles represent titers below the threshold, and crossed circles represent titers above the threshold.

[0126] Figure 3. Effect of dasatinib treatment on anti-AAV8 IgG titers. Serum was isolated from blood collected on study days 0, 8, 15, 21, 29, and 36 ( Figure 1 ), and anti-AAV8 IgG was measured by ELISA performed on immobilized empty AAV8 capsids. IgG antibody titers were determined by ELISA OD values ​​of serial dilutions (1:10 and 7 steps of serial dilution 1:3). To determine the average background content, readings were pooled from all mouse groups on day 0. The positive threshold is represented by the dotted line. The kinetics of anti-AAV8 IgG antibody titers are shown for each mouse group ( Figures 3A-3C ). Open circles represent titers below the threshold, and crossed circles represent titers above the threshold.

[0127] Figure 4. Dasatinib treatment allows transgene expression after rAAV re-administration. Serum was isolated from blood collected on study days 0, 8, 15, 21, 29, and 36 ( Figure 1 ). Figure 4A ) The expression of the first transgenic hSEAP was measured by chemiluminescence. Figure 4B ) After AAV8 re-administration, expression of the second transgene hFactorIX was measured by ELISA. Mean concentrations are shown + / - SD.

[0128] Figure 5. Effect of dasatinib on AAV-mediated cytokine release in human whole blood. After incubation with 5E11 vg / mL of AAV8-hSEAP for 24 hours, low levels of IFN-γ and IL-6 production in whole blood were stimulated. In the presence of dasatinib at 12.5 or 50 nM, this production was reduced in a dose-dependent manner. Cytokine concentrations (pg / mL+ / -SD, left), fold increases relative to the levels measured in PBS controls (middle graph), and percentages of inhibition of AAV-dependent cytokine release (right graph, calculated as 100x (concentration of AAV8 alone vs. concentration of AAV8 and dasatinib) / (concentration of AAV8 alone) are depicted, respectively. Figure 5A ) Interferon-γ production. Figure 5B )IL-6 production.

[0129] Figure 6: Dasatinib inhibits AAV8-dependent cytokine release in human whole blood. Fresh whole blood from 4 healthy donors was incubated with PBS or AAV8-hSEAP (5e11 vg / mL) with IVIG (PRIVIGEN, 1 / 100) for 24 hours. Dasatinib was added at a concentration of 50 nM 1 hour before the addition of AAV8. In some culture wells, a second dose of dasatinib (dasa 50 nM 2x) was added 9 hours after AAV8 treatment. After 24 hours, plasma was collected and cytokines were measured using Quanterix kits. Responses to IFN-γ and TNF-α ( Fig. 6A ), IL-6 and IL-1α ( Figure 6B ) and IL-2 and IL-1β ( Figure 6C ) fold increase in cytokines relative to PBS, and percent inhibition in the presence of dasatinib (mean + / - SEM).

[0130] Figure 7 : In vivo study to evaluate the effects of extended dasatinib treatment. All mouse groups except Group 1 were immunized with intravenous AAV8-hSeap (1e12 vg / kg) on ​​day 1. Four groups received intravenous AAV8-FactorIX (3e13 vg / kg) on ​​day 43. Groups 3 and 4 received dasatinib treatment for 1 or 2 weeks, respectively, at the same time as the first AAV administration. Dasatinib was administered at a dose of 50 mg / kg per gavage, twice a day, and started 1 hour before the first AAV treatment.

[0131] Figure 8: Extending dasatinib treatment from 1 to 2 weeks improves the inhibition of IgM formation against AAV8. Fig. 8A and Figure 8BIn the experiment described in , anti-AAV8 IgM titers (median + / - SD) were measured in each group of mice from day 0 (before AAV8-hSeap administration) to day 64, as described for Figure 2. Each circle represents an individual IgM titer.

[0132] Figure 9: Extending dasatinib treatment from 1 to 2 weeks improves the inhibition of IgG formation against AAV8. Fig.9A and Fig. 9B In the experiment described in , anti-AAV8 IgG titers (median + / - SD) were measured in each group of mice from day 0 (before AAV8-hSeap administration) to day 64, as described for Figure 3. Each circle represents an individual IgG titer.

[0133] Fig.10 : Extended dasatinib treatment after first AAV8 administration improves transgene expression after re-administration. Fig.10 In the experiments described in , the levels of hFIX expression (ng / mL, median + / - SD) were measured for each group starting on day 42 (one day before AAV8-hFIX administration) and continuing until day 64.

[0134] Fig.11 : Dasatinib treatment inhibits cytokine and chemokine release by mouse splenocytes in response to AAV8. Splenocytes were isolated from C57 / Bl6 mice and incubated with AAV8-hSeap at MOI 1E5 for 24 hours in the presence or absence of dasatinib (100nM, 50nM or 12.5nM). Cytokines and chemokines were measured in culture supernatants. LPS (positive) and PBS (negative) controls are shown in the figure. Mean + / - SD (triplicate). Dashed line: lower limit of quantitation.

[0135] Figure 12: Dasatinib treatment reduces T cell responses to AAV2 and AAV9 in vitro. PBMCs from healthy human donors were stimulated with a peptide pool covering AAV2 and AAV9 capsid sequences in the absence (black bars) or presence of 100 nM dasatinib (grey bars). After 48 hours, IFN-γ- and TNF-α-secreting cells were measured by Fluorospot. Mean spot-forming cells + / - SD are shown for 1E6 PBMCs. Dashed line: positive threshold.

[0136] Examples

[0137] The following are examples of methods and compositions of the present invention. It will be appreciated that various other aspects may be implemented, given the general description given above.

[0138] Example 1. Dasatinib prevents anti-AAV8 antibody formation after rAAV8 administration in mice.

[0139] To assess whether dasatinib can effectively prevent anti-AAV antibody formation after rAAV administration and allow efficient re-administration of rAAV, studies were performed in mice ( Figure 1 ). On study day 1, the C57Bl / 6 mouse group was intravenously administered a first rAAV8 encoding hSEAP (human secretory alkaline phosphatase, under the control of the CMV promoter) at a dose of 1E12 vg / kg. On study day 22, they were administered a second rAAV8 encoding hFIX (human factor IX, under the control of the CMV promoter) at a dose of 5E13 vg / kg. The unimmunized control group 1 did not receive the first rAAV8 injection, while the immunized control group 2 received two injections. Group 3 mice were treated with dasatinib by gavage twice a day at a dose of 50 mg / kg for 7 days starting on the day of the first rAAV8 administration. Blood samples were collected on study days 0 (one day before the first AAV administration), 8, 15, 21, 29 and 36. Serum IgM and IgG against the AAV8 capsid were titrated in serum samples (Figures 2 and 3). One week after the first rAAV8 administration, a peak of anti-AAV8 IgM was observed on day 8 for immunized control group 2 and on day 29 for unimmunized control group 1 (10 / 10 mice were above the threshold) (Figure 2). In group 3 treated with dasatinib, sera from all mice remained below the IgM positive threshold until the second rAAV8 was administered. The IgM positivity of mouse 302, which only appeared on day 15, was attributed to technical problems in the ELISA. Anti-AAV8 IgG titers were significantly reduced in group 3 treated with dasatinib compared to immunized control group 2. On study day 21, only 4 / 10 group 3 mice were above the positive threshold, while 10 / 10 group 2 mice were above this threshold (Figure 3). On day 36, two weeks after rAAV8 was re-administered, anti-AAV8 IgG titers were comparable in all mouse groups, indicating that the effect of dasatinib was transient and did not affect subsequent immune responses to rAAV8. These data indicate that dasatinib efficiently reduces anti-AAV8 antibody formation following rAAV8 administration in mice.

[0140] Example 2. Brief dasatinib treatment allows transgene expression following rAAV8 re-administration.

[0141] The expression of the first transgene hSEAP and the second transgene hFIX was measured in serum samples from the above experiments ( Figure 1 , Example 1). As expected, human SEAP was detected in the sera of mice from Group 2 and Group 3 7 days after injection of AAV8-hSEAP on Study Day 1 ( Figure 4AAfter the second administration of rAAV8 on study day 22, hFIX transgene expression could not be detected in the sera from mice in immunized control group 2 ( Figure 4B ), and remained at baseline until day 36 (14 days after AAV8-hFIX administration), indicating that re-administration was completely ineffective. In contrast, hFIX expression was detected in both: serum from all mice in unimmunized group 1 (mean: 2107 ng / ml) and from most mice in group 3 treated with dasatinib (mean: 1398 ng / ml). This data suggests that dasatinib treatment at the time of the first rAAV administration at least partially restored the efficacy of transduction by the second rAAV. In summary, the results from Examples 1 and 2 indicate that the inhibition of anti-AAV8 antibody formation caused by brief dasatinib treatment allows efficient AAV re-administration.

[0142] Example 3. Dasatinib dose-dependently inhibits AAV-induced cytokine production in human blood.

[0143] To assess whether dasatinib affects AAV-mediated cytokine release, assays were performed in whole blood from healthy donors. This donor was seropositive for anti-AAV8 antibodies (IgG titer: 1 / 21870; IgM titer: 1 / 810). Whole blood from AAV8 pre-immune donors was incubated in triplicate with 5E11 vg / mL (GC / mL) of AAV8-hSEAP vector in the presence or absence of 12.5 nM or 50 nM dasatinib. Lipopolysaccharide (LPS) and Lemtrada (alemtuzumab, anti-CD52, Genzyme), a monoclonal antibody known to induce strong cytokine release in the blood, were used as positive controls at concentrations of 1 μg / ml and 0.1 μg / ml, respectively. PBS was used as a negative control and for assessing baseline cytokine production. Dasatinib was added to the blood 1 hour before incubation with AAV8-hSEAP. After 24 hours, plasma supernatants were collected and cytokines were measured using Quanterix kits with the SP-X imaging and analysis system (Simoa). Figure 5A and 5B It was shown that after 24 hours of incubation with 5E11 vg / mL of AAV8-hSEAP, low levels of IFN-γ and IL-6 production in whole blood were stimulated. This production was reduced in a dose-dependent manner in the presence of dasatinib at 12.5 or 50 nM. These results suggest that inhibition of cytokine release may be the mechanism by which dasatinib inhibits the production of anti-AAV antibodies.

[0144] Example 4. Dasatinib dose-dependently inhibits AAV-induced cytokine production in human blood.

[0145] In order to study the effect of dasatinib on AAV-dependent cytokine release in more detail, another whole blood assay was performed. Whole blood from four healthy donors was incubated with AAV8-hSEAP (5e11vg / ml) for 24 hours. Intravenous immunoglobulin (IVIG, Privigen) containing anti-AAV antibodies was added to the blood sample. 1 hour before AAV treatment, dasatinib was added at 50nM, and PBS was used as a negative control. The half-life of dasatinib is very short (Lindauer M et al., Recent Results Cancer Res. 2010; 184: 83-102), and dasatinib was added for the second time 9 hours after AAV treatment. After 24 hours, plasma supernatant was collected and cytokines were measured using the Quanterix kit. Figure 6 shows that in the presence of dasatinib, the production of IFN-γ, IL-6, IL-2, TNF-α, IL-1α and IL-1β is inhibited. This inhibition was more pronounced when dasatinib was added 1 hour before and 9 hours after AAV treatment. These results indicate that dasatinib inhibits the release of multiple proinflammatory cytokines (including IL-6 and IL-1β), which have been reported to stimulate antibody responses to AAV capsids (Kuranda K et al., J Clin Invest. 2018 128(12):5267-5279). Inhibition of AAV-mediated cytokine release may contribute to the effect of dasatinib on antibody formation.

[0146] Example 5. Prolonged dasatinib treatment improves anti-AAV8 antibody suppression after rAAV8 administration and allows efficient re-administration in mice.

[0147] In the experiments described in Example 1, although a strong inhibition of anti-AAV antibody formation was observed in most mice, 4 of 10 animals treated with dasatinib began to produce anti-AAV8 IgG before re-dosing ( Figure 3C , days 15-21). After cessation of dasatinib treatment, the presence of residual AAV8 particles in the circulation and in tissues may lead to this antibody formation. For this reason, we investigated whether prolonged dasatinib treatment could more efficiently suppress the humoral response to the AAV8 capsid. For this purpose, the same regimen as in Example 1 was applied, but with 1 or 2 weeks of dasatinib treatment. In addition, re-dosing was performed at a later time point (day 43 instead of day 22) in order to be able to observe potential antibody formation after cessation of dasatinib treatment. The regimen is depicted in Figure 7 middle.

[0148] Circulating IgM and IgG against AAV8 capsid were titrated in serum samples (Figures 8 and 9). One week after the first rAAV8 administration, a peak of anti-AAV8 IgM was observed on day 8 for immunized control group 2 and on day 50 for unimmunized control group 1 (10 / 10 mice above threshold) (Figure 8). In group 3, treated with dasatinib for 1 week, IgM titers remained low to negative until the second rAAV8 was administered. On day 42 (before AAV8 was re-administered), four of 15 mice had positive IgM titers against AAV8. Group 4, treated with dasatinib for 2 weeks, had a stronger inhibition of IgM production. In this group, only one mouse showed a positive IgM titer on day 42.

[0149] In group 3 treated with dasatinib for 1 week, 3 / 15 mice showed negative anti-AAV8 IgG titers on day 42, while all 15 mice in immunized control group 2 had very high IgG titers (Figure 9). As for IgM, the inhibition of IgG formation was stronger in group 4 mice treated with dasatinib for 2 weeks. In this group, 5 / 15 mice had IgG titers below the positive threshold on day 42, and the median titer was much lower than that in group 3. In summary, extending dasatinib treatment from 1 week to 2 weeks produced more effective inhibition of IgM and IgG formation against AAV8.

[0150] On day 64, 3 weeks after rAAV8 re-administration, anti-AAV8 IgG titers were comparable in all mouse groups, again indicating that the effect of dasatinib was transient and did not affect subsequent immune responses to rAAV8.

[0151] Inhibition of antibody formation to AAV8 correlated with improved hFIX transgene expression after re-administration, indicating efficient transduction by the second AAV8 ( Fig.10 hFIX expression was negatively correlated with IgG and IgM titers measured on day 42, prior to re-dosing. In mice that remained IgG negative on day 42, hFIX levels on day 64 were within the range of those measured in the unimmunized control group 1. hFIX expression was higher in group 4, treated with dasatinib for 2 weeks, compared to group 3, which was treated for only 1 week, reflecting a more complete inhibition of anti-AAV8 antibody formation.

[0152] Example 6: Dasatinib reduces AAV8-dependent cytokine and chemokine release in mouse splenocytes.

[0153] To investigate the mechanism of dasatinib-mediated inhibition of humoral responses to AAV8 observed in mice (Examples 1 and 5), we investigated whether the rapid production of cytokines and chemokines by immune cells was affected by dasatinib treatment. Total splenocytes from C57BL / 6 mice were cultured for 24 h in the presence or absence of different concentrations of dasatinib. The secreted cytokines and chemokines in the culture supernatants were analyzed using the ProcartaPlex immunoassay (Luminex). Fig.11 As shown, dose-dependent reductions were measured for a range of cytokines (IL-6, TNF-α) and chemokines (IP-10 / CXCL10, MCP-1, MCP-3, MIP-1α, MIP-1β, MIP-2α) that are characteristic of the early innate immune response to rAAV (Shirley JL et al. Mol Ther. 2020 Mar 4;28(3):709-722).

[0154] Together with Examples 3 and 4, which illustrate the effects of dasatinib on cytokine production by human PBMCs, these results show that dasatinib downregulates the innate immune response to AAV in both humans and mice. They also suggest that the inhibition of antibody responses to AAV vectors observed in mice may be at least in part a result of this inhibition of early cytokine and chemokine production. In particular, IL-6 release has been shown to contribute to anti-AAV antibody formation (Kuranda K et al., J Clin Invest. 2018 Dec 3; 128(12): 5267-5279).

[0155] Example 7. Dasatinib inhibits human T cell responses to AAV2 and AAV9 in vitro.

[0156] The above examples show that dasatinib treatment has the potential to suppress both innate and humoral immune responses to AAV8. An important question is its potential impact on cellular adaptive immune responses. It has been reported that T cell responses to rAAV can induce clearance of AAV-transduced cells, thereby shortening the duration of transgene expression. It is also known clinically that T cell responses to AAV can mediate liver toxicity.

[0157] T cell responses to AAV can be measured in healthy human blood donors that have been previously infected with wild-type AAV. FluoroSpot assays reveal the ratio of PBMCs that produce IFN-γ and TNF-α induced after incubation with a pool of AAV capsid peptides. We used this assay to assess the response of PBMCs from two healthy blood donors to three different peptide pools covering the capsid sequences of AAV2 and AAV9 (Figure 12). In the absence of dasatinib, PBMCs from donor 1 showed a positive IFN-γ response to AAV9 pool 1, and a positive TNF-α response to AAV2 pool 2 and AAV9 pools 2 and 3. Donor 2 had positive IFN-γ and / or TNF-α responses to AAV9 pools 2 and 3. All of these reactions were inhibited in the presence of 100nM dasatinib.

[0158] These results suggest that dasatinib has the potential to block both aspects of the adaptive immune response to AAV capsids, namely the T cell response and the antibody response. This makes the compound promising for highly effective reduction of the immunogenicity of AAV-based gene therapy vectors. Importantly, the inhibitory effect of dasatinib is not limited to the immune response to the AAV8 serotype, as T cell responses to AAV2 and AAV9 were also suppressed by dasatinib treatment.

[0159] ***

[0160] Although the present invention has been previously described in considerable detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A recombinant viral vector comprising a heterologous polynucleotide for use in the treatment of a disease in an individual, wherein the treatment comprises (a) administering the recombinant viral vector to the individual, and (b) administering a tyrosine kinase inhibitor (TKI) to the individual for preventing or reducing the formation of anti-drug antibodies (ADA) associated with the administration of the recombinant viral vector.

2. Use of a recombinant viral vector comprising a heterologous polynucleotide in the manufacture of a medicament for treating a disease in an individual, wherein the treatment comprises (a) administering the recombinant viral vector to the individual, and (b) administering a tyrosine kinase inhibitor (TKI) to the individual for preventing or reducing the formation of anti-drug antibodies (ADA) associated with the administration of the recombinant viral vector.

3. A method for treating a disease in an individual, wherein the method comprises (a) administering to the individual a recombinant viral vector comprising a heterologous polynucleotide, and (b) administering a tyrosine kinase inhibitor (TKI) to the individual for preventing or reducing the formation of anti-drug antibodies (ADA) associated with the administration of the recombinant viral vector.

4. A tyrosine kinase inhibitor (TKI) for use in preventing or reducing the formation of anti-drug antibodies (ADA) associated with administration of a recombinant viral vector comprising a heterologous polynucleotide to an individual.

5. Use of a tyrosine kinase inhibitor (TKI) in the manufacture of a medicament for preventing or reducing the formation of anti-drug antibodies (ADA) associated with the administration of a recombinant viral vector comprising a heterologous polynucleotide to an individual.

6. A method for preventing or reducing the formation of anti-drug antibodies (ADA) associated with administering a recombinant viral vector comprising a heterologous polynucleotide to an individual, the method comprising administering a tyrosine kinase inhibitor (TKI) to the individual.

7. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the TKI is a Lck and / or Src kinase inhibitor, in particular dasatinib.

8. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein (administration of) the TKI results in (i) inhibiting the formation of ADA bound to the recombinant viral vector, (ii) inhibiting the activation of T cells (induced by the recombinant viral vector), (iii) inhibiting the cytotoxic activity of T cells (induced by the recombinant viral vector), (iv) inhibiting the activation of B cells (induced by the recombinant viral vector) (v) inhibiting the formation of plasma cells (induced by the recombinant viral vector), and / or (vi) inhibiting the secretion of cytokines by immune cells (induced by the recombinant viral vector), particularly wherein the cytokines are one or more cytokines selected from the group consisting of: IL-2, TNF-α, IFN-γ, IL-6 and IL-1β.

9. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein (administration of) the TKI results in a reduction in serum levels of one or more cytokines in the individual, particularly wherein the one or more cytokines are selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6 and IL-1β.

10. The recombinant viral vector, TKI, use or method according to any of the preceding claims, wherein the TKI is administered (i) before, simultaneously with or after the administration of the recombinant viral vector, (ii) intermittently or continuously, and / or (iii) orally.

11. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the TKI is administered at a dose sufficient to (i) inhibiting the formation of ADA bound to the recombinant viral vector, (ii) inhibiting the activation of T cells (induced by the recombinant viral vector), (iii) inhibiting the cytotoxic activity of T cells (induced by the recombinant viral vector), (iv) inhibiting the activation of B cells (induced by the recombinant viral vector) (v) inhibiting the formation of plasma cells (induced by the recombinant viral vector), and / or (vi) inhibiting the secretion of cytokines by immune cells (induced by the recombinant viral vector), particularly wherein the cytokines are one or more cytokines selected from the group consisting of: IL-2, TNF-α, IFN-γ, IL-6 and IL-1β.

12. The recombinant viral vector, TKI, use or method of any preceding claim, wherein the TKI is administered at a dose sufficient to reduce serum levels of one or more cytokines in the individual.

13. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the TKI is administered at a dose sufficient to reduce the secretion of one or more cytokines by immune cells in the individual.

14. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the TKI is administered at an effective dose.

15. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the TKI is administered at a dose of about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg or 200 mg, particularly at a dose of about 100 mg or less.

16. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein administration of the TKI continues for a period of time during which ADA formation is expected, and / or is stopped after the formation of ADA is prevented or reduced.

17. A recombinant viral vector, TKI, use or method according to any of the preceding claims, wherein the administration of the TKI is associated with the first administration of the recombinant viral vector, and is optionally performed before, simultaneously with or after the first administration of the recombinant viral vector.

18. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the administration of the recombinant viral vector (i) in an effective dose, (ii) is administered parenterally, in particular intravenously, and / or (iii) is the first administration of the recombinant viral vector to the individual.

19. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the recombinant viral vector comprises a lentiviral vector, an adenoviral vector or an adeno-associated (AAV) vector.

20. The recombinant viral vector, TKI, use or method according to claim 19, wherein the recombinant lentiviral vector comprises an envelope protein that binds to the ADA.

21. The recombinant viral vector, TKI, use or method of claim 19, wherein the recombinant AAV vector comprises a capsid protein that binds to the ADA.

22. The recombinant viral vector, TKI, use or method according to claim 19 or 21, wherein the AAV vector comprises VP1, VP2 and / or VP3 capsid proteins that bind to the ADA.

23. The recombinant viral vector, TKI, use or method of claim 19, 21 or 22, wherein the AAV vector comprises a VP1, VP2 and / or VP3 capsid protein having 70% or higher sequence identity with a VP1, VP2 and / or VP3 capsid protein selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8 VP1, VP2 and / or VP3 capsid protein.

24. A recombinant viral vector, TKI, use or method according to claim 19, 21 or 22, wherein the AAV vector comprises VP1, VP2 and / or VP3 capsid proteins having 100% sequence identity with VP1, VP2 and / or VP3 capsid proteins selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8 VP1, VP2 and / or VP3 capsid proteins.

25. The recombinant viral vector, TKI, use or method of any one of the preceding claims, wherein the individual is at risk of developing ADA that binds to the recombinant viral vector.

26. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the individual is free of ADA bound to the recombinant viral vector before and / or after administration of the TKI.

27. The recombinant viral vector, TKI, use or method of any one of the preceding claims, wherein the individual is at risk of developing ADA to the polypeptide encoded by the heterologous polynucleotide.

28. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein the ADA comprises IgG, IgM, IgA, IgD and / or IgE, in particular wherein the ADA comprises IgG and / or IgM.

29. A recombinant viral vector, TKI, use or method according to any of the preceding claims, wherein the ADA bound to the recombinant viral vector is reduced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, optionally compared to natural history data of a relevant control group not administered with the TKI.

30. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein transgene expression is increased upon re-administration of the viral vector, in particular compared to transgene expression before re-administration of the viral vector.

31. The recombinant viral vector, TKI, use or method of any of the preceding claims, wherein transgene expression is increased by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to transgene expression prior to re-administration of the viral vector.

32. The recombinant viral vector, TKI, use or method according to any one of the preceding claims, wherein transgene expression is maintained upon re-administration of the viral vector, in particular compared to transgene expression before re-administration of the viral vector.

33. The invention as hereinbefore described.

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