Preventing or mitigating adverse effects associated with recombinant viral vectors

Abstract

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

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the prevention or mitigation of adverse effects associated with gene therapy, such as the formation of anti-drug antibodies. In particular, the present invention relates to the prevention or mitigation of such adverse effects using tyrosine kinase inhibitors, such as dasatinib. [Background technology]

[0002] background Recombinant adeno-associated viruses (rAAV) or AAV vectors are viral vectors used in in vivo gene therapy to deliver therapeutic transgenes to target cells. rAAV-mediated gene therapy is highly promising for a large range of genetic diseases. Recombinant AAV capsids are generally derived from wild-type AAVs, which naturally infect specific cell types depending on their serotype. Recombinant AAV-mediated transgene delivery allows for long-term expression of therapeutic proteins (Nathwani, A. C. 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 against the viral capsid and, in some cases, against the transgene product (Ronzitti, G. et al. Human immune responses to Adeno-Associated Virus (AAV) vectors. Frontiers in Immunology 11:670 (2020) (Non-Patent Document 2); Shirley, J. Let al. Immune responses to viral gene therapy vectors. Molecular Therapy 28:709-722 (2020) (Non-Patent Document 3)). Innate and adaptive immune responses can result in cytokine release, complement activation, and cytotoxic T cell responses, but most commonly, they can result in the formation of antibodies against the AAV capsid. This humoral response is characterized by the secretion of anti-AAV IgM and IgG. These antibodies are mostly neutralizing antibodies that prevent further rAAV treatment. Furthermore, they may cause some of the toxicities observed in clinical trials by mediating complement activation.For these reasons, strategies to mitigate antibody formation against rAAV are highly needed (Verdera HC et al., AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Molecular Therapy (2020) (Non-Patent Document 4)).

[0003] The tyrosine kinase inhibitor dasatinib was identified as a potent compound that turned off cytokine release and T cell activation in mice treated with a T cell bispecific antibody (Leclercq et al., Journal for ImmunoTherapy of Cancer, 2021;9(7) (Non-Patent Document 5)). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nathwani,ACet al.Long-term safety and efficacy of factor IX gene therapy in hemophilia B.New England Journal of Medicine 371:1994-2004(2014) [Non-patent document 2] Ronzitti,G.et al.Human Immune responses to Adeno-Associated Virus(AAV)vectors.Frontiers in Immunology 11:670(2020) [Non-patent document 3] Shirley,JLet al.Immune responses to viral gene therapy vectors.Molecular Therapy 28:709-722(2020) [Non-patent document 4] Verdera HCet al.,AAV Vector Immunogenicity in Humans:A Long Journey to Successful Gene Transfer.Molecular Therapy(2020) [Non-Patent Document 5] Leclercq et al.,Journal for ImmunoTherapy of Cancer,2021;9(7) Summary of the Invention

[0005] Description of the Invention The present inventors have found that tyrosine kinase inhibitors, particularly dasatinib, can be used to prevent the formation of anti-drug antibodies (ADA) induced by recombinant viral vector-based gene therapy.

[0006] Using an in vivo model of recombinant viral vector-based gene therapy, specifically AAV-based delivery of human proteins (hSEAP, hFactorIX) in mice, we evaluated the effect of dasatinib on the (unwanted) formation of anti-drug antibodies (ADAs) associated with the administration of recombinant viral vectors. Mice were intravenously administered a first rAAV8 encoding hSEAP and then re-administered a second rAAV8 encoding hFactorIX. Blood samples were collected and analyzed for the presence of ADAs and transgene expression. We demonstrate that dasatinib can effectively reduce ADA formation after in vivo administration of recombinant AAV (rAAV). Furthermore, we demonstrate that dasatinib enables re-administration of rAAV of the same serotype. These effects can be achieved at clinically relevant dasatinib concentrations. We propose that co-administration of dasatinib with recombinant viral vectors prevents ADA formation against recombinant viral vectors. This invention is broadly applicable to the enhancement of gene therapy treatments. For example, overcoming ADA against the AAV capsid has the potential to allow repeat administration of AAV gene therapy products to patients previously administered where efficacy levels were not achieved or were lost due to time or other confounding issues.

[0007] Thus, 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, said treatment comprising: (a) administering a recombinant viral vector to an individual; and (b) administering a tyrosine kinase inhibitor (TKI) to the individual to prevent or reduce the formation of anti-drug antibodies (ADA) associated with administration of the recombinant viral vector. The present invention provides a recombinant viral vector comprising:

[0008] The present invention further provides the use of a recombinant viral vector comprising a heterologous polynucleotide in the manufacture of a medicament for the treatment of a disease in an individual, said treatment comprising: (a) administering a recombinant viral vector to an individual; and (b) administering a tyrosine kinase inhibitor (TKI) to the individual to prevent or reduce the formation of anti-drug antibodies (ADA) associated with administration of the recombinant viral vector. Provide for use, including

[0009] The present invention also provides a method for the treatment of a disease in an individual, said method comprising: (a) administering to an individual a recombinant viral vector comprising a heterologous polynucleotide; and (b) administering a tyrosine kinase inhibitor (TKI) to the individual to prevent or reduce the formation of anti-drug antibodies (ADA) associated with administration of the recombinant viral vector. The present invention provides a method comprising:

[0010] 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 the administration of a recombinant viral vector comprising a heterologous polynucleotide to an individual.

[0011] 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.

[0012] The present invention also provides a method for preventing or mitigating the formation of anti-drug antibodies (ADA) associated with the administration of a recombinant viral vector comprising a heterologous polynucleotide to an individual, the method comprising administering a tyrosine kinase inhibitor (TKI) to the individual.

[0013] Unless otherwise defined herein, terms are used herein as commonly used in the art.

[0014] In some embodiments, the TKI is an Lck and / or Src kinase inhibitor. In more specific embodiments, the TKI is dasatinib.

[0015] "Dasatinib" is a tyrosine kinase inhibitor (TKI). It is sold under the trade name Sprycel® (among other things) 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.

[0016] CAS number: 302962-49-8 IUPAC name: N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate Chemical structure: TIFF2025526697000001.tif26128

[0017] In some embodiments, the administration of a TKI causes inhibition of the formation of anti-drug antibodies (ADA) that bind to the recombinant viral vector.

[0018] "Anti-drug antibodies" or "ADAs" refer to antibodies that bind to therapeutic agents (e.g., AAV capsid proteins) and can affect an individual's serum concentration and function of the therapeutic agent. The presence of ADAs can increase the clearance of the therapeutic agent through the formation of immune complexes between the therapeutic agent and antibodies (neutralizing, non-neutralizing, or both) and shorten the half-life of the therapeutic agent. Furthermore, the activity and efficacy of the therapeutic agent (e.g., its ability to transduce target cells) can be reduced by antibody binding to the therapeutic agent (especially in the case of neutralizing ADAs). ADAs can also be associated with allergic or hypersensitivity reactions and other adverse events.

[0019] "Anti-drug antibody formation" or "ADA formation" refers to an immunological, particularly humoral, response in an individual's body triggered by the application of a therapeutic agent (e.g., an AAV-based recombinant viral vector). Such a response may include cellular responses of T cells, particularly CD4+ T cells, such as proliferation, differentiation, cytokine secretion, and / or expression of activation markers. Additionally, such a response may include B cell activation, formation of plasma cells, formation of memory B cells, and / or shedding of antibodies (such as ADAs).

[0020] In some embodiments, the administration of a TKI causes inhibition of T cell activation (induced by the recombinant viral vector).

[0021] As used herein, "T cell activation" or "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly CD4+ or CD8+ T cells, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein. In certain embodiments, T cell activation is determined by measuring the expression of CD25 and / or CD69 on T cells, e.g., by flow cytometry.

[0022] In some embodiments, the administration of the TKI causes inhibition of T cell proliferation (induced by the recombinant viral vector).

[0023] In some embodiments, administration of a TKI causes inhibition of the cytotoxic activity of T cells (e.g., against cells expressing a polypeptide or peptide encoded by a heterologous polynucleotide introduced into the cells by a recombinant viral vector).

[0024] "Cytotoxic activity" of T cells refers to the induction of cell lysis (i.e., killing) by T lymphocytes, particularly CD8+ T cells. Cytotoxic activity typically involves degranulation of T lymphocytes, which is associated with the release of cytotoxic effector molecules such as granzyme B and / or perforin from the T lymphocytes.

[0025] In some embodiments, the (administration of) the TKI causes inhibition of T cell receptor signaling in T cells (induced by the recombinant viral vector).

[0026] "T cell receptor signaling" refers to the activity of signaling pathways downstream of the T cell receptor (TCR) in T lymphocytes following engagement of the TCR, including signaling molecules including tyrosine kinases such as Lck kinase.

[0027] In some embodiments, the administration of the TKI causes inhibition of B cell activation (induced by the recombinant viral vector).

[0028] "B cell activation" refers to one or more cellular responses of B lymphocytes, particularly naive or memory B cells, selected from proliferation, differentiation (particularly 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 certain embodiments, B cell activation is determined by measuring the expression of CD69 on B cells, e.g., by flow cytometry.

[0029] In some embodiments, administration of a TKI causes inhibition of cytokine secretion by immune cells (induced by the recombinant viral vector). In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6, and IL-1β. In some embodiments, the immune cells are myeloid cells, CD8+ T cells, or CD4+ cells.

[0030] In some embodiments, the inhibition is reversible (i.e., the inhibition can be reversed so that the level of the inhibited parameter returns to a level similar to that before the inhibition). In some embodiments, the inhibition is reversed after the TKI has not been administered to the individual for a given period of time (i.e., after administration of the TKI has ceased). In some embodiments, the 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.

[0031] The inhibition can be partial or complete, hi some embodiments, the inhibition is clinically meaningful and / or statistically significant.

[0032] In some embodiments, administration of a TKI causes a decrease in serum levels of one or more cytokines in an individual. In some embodiments, the one or more cytokines are selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6, and IL-1β. In some embodiments, the decrease is sustained after the TKI has not been administered to the individual for a given amount of time. In some embodiments, 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 decrease in serum levels is particularly when compared to the serum levels of an individual (including the same individual) not administered the TKI (i.e., in such cases, the serum levels are decreased compared to serum levels without / before administration of the TKI). Said reduction in serum levels is particularly when compared to the serum levels of an individual (including the same individual) who has been administered a recombinant viral vector (particularly the first administration) but not a TKI (i.e., in such cases, the serum levels are reduced compared to the serum levels with / after administration of the recombinant viral vector but without / before administration of the TKI). Without said reduction, serum levels and / or cytokine secretion may be elevated / increased, particularly in association with (administration of) a recombinant viral vector. In some embodiments, said reduction is clinically meaningful and / or statistically significant.

[0033] In some embodiments, the TKI is administered upon clinical manifestation of increased serum levels of one or more cytokines. In some embodiments, the one or more cytokines are selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6, and IL-1β. The administration 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 after the manifestation of increased serum levels of one or more cytokines (i.e., the onset of clinical symptoms such as fever). In some embodiments, the TKI is administered in response to clinical manifestation of increased serum levels of one or more cytokines (in an individual).

[0034] In some embodiments, the administration of the TKI is before the administration of the recombinant viral vector. In some embodiments, the administration of the TKI is simultaneous with the administration of the recombinant viral vector. In some embodiments, the administration of the TKI is after the administration of the recombinant viral vector. When the administration of the TKI is before or after the administration of the recombinant viral vector, the administration of such 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 or 24 hours before or after the administration of the recombinant viral vector, respectively. The administration of the TKI can be intermittent or continuous. In some embodiments, the administration of the TKI is oral.

[0035] In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of the adverse effects of the recombinant viral vector. An "adverse effect," also sometimes referred to as a "side effect" or "adverse event" (particularly in clinical trials), herein refers to a harmful and undesirable effect resulting from drug therapy, particularly in the treatment of an individual with a recombinant viral vector. In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of the formation of ADAs that bind to the recombinant viral vector. In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of T cell activation (induced by the recombinant viral vector). In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of T cell cytotoxic activity (induced by the recombinant viral vector). In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of T cell receptor signaling in T cells (induced by the recombinant viral vector). In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of cytokine secretion by immune cells (induced by the recombinant viral vector). In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6, and IL-1β. In some embodiments, the immune cells are myeloid cells, CD8+ T cells, or CD4+ cells. The inhibition can be partial or complete. In some embodiments, the inhibition is clinically meaningful and / or statistically significant.

[0036] In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of antibody production (e.g., ADA) by B cells (induced by the recombinant viral vector). In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of B cell activation (induced by the recombinant viral vector). In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of B cell differentiation (induced by the recombinant viral vector). In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of plasma cell formation (induced by the recombinant viral vector). In some embodiments, the TKI is administered at a dose sufficient to cause inhibition of cytokine secretion by B cells (induced by the recombinant viral vector). In some embodiments, the cytokine is one or more cytokines selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-4, IL-6, and GM-CSF. The inhibition can be partial or complete. In some embodiments, the inhibition is clinically meaningful and / or statistically significant.

[0037] Said reduction in serum levels or cytokine secretion is particularly when compared to the serum levels or cytokine secretion of an individual (including the same individual) who has not been administered a TKI (i.e., in such cases, the serum levels are reduced compared to the serum levels without / before administration of the TKI). Said reduction in serum levels or cytokine secretion is particularly when compared to the serum levels or cytokine secretion of an individual (including the same individual) who has been administered a recombinant viral vector (particularly the first administration) but not a TKI (i.e., in such cases, the serum levels are reduced compared to the serum levels with / after administration of the recombinant viral vector but without / before administration of the TKI). Without said reduction, serum levels and / or cytokine secretion may be elevated / increased, particularly in association with (administration of) a recombinant viral vector. In some embodiments, said reduction is clinically meaningful and / or statistically significant. Said inhibition may be partial or complete. In some embodiments, said inhibition is clinically meaningful and / or statistically significant.

[0038] In some embodiments, the administration of the TKI is at an effective dose.

[0039] An "effective amount" or "effective dose" of an agent, e.g., a TKI or viral vector, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0040] In some embodiments, 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. In some embodiments, the TKI is administered at a dose of about 20 mg. In some embodiments, the TKI is administered at a dose of about 70 mg. In some embodiments, the TKI is administered at a dose of about 80 mg. In some embodiments, the TKI is administered at a dose of about 100 mg. In some embodiments, the TKI is administered at a dose of about 140 mg.

[0041] In some embodiments, the TKI is administered at a dose of about 100 mg or less. In some embodiments, the TKI is administered at a dose of about 20 mg. In some embodiments, the TKI is administered at a dose of about 70 mg. In some embodiments, the TKI is administered at a dose of about 80 mg. In some embodiments, the TKI is administered at a dose of about 100 mg.

[0042] In some embodiments, the TKI is administered daily. In some embodiments, the TKI is administered once a day. In some embodiments, the TKI is administered at a dose of about 100 mg once a day. In some embodiments, the TKI is administered for the duration of the adverse effect (i.e., the TKI is administered from the onset of the adverse effect until the adverse effect is reduced or eliminated). In some embodiments, the TKI is stopped after preventing or reducing the formation of ADA. In some embodiments, the TKI is stopped after reducing ADA. The reduction is particularly clinically meaningful and / or statistically significant. In some embodiments, the TKI is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times during the course of treating an individual with a recombinant viral vector. In some embodiments, the TKI is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the TKI is administered 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 embodiments, the TKI is administered concomitantly with the first administration of a recombinant viral vector. The first administration is particularly the first administration of a recombinant viral vector in the course of treating an individual with a recombinant viral vector. In some embodiments, the TKI is administered simultaneously with the first administration of a recombinant viral vector. In some embodiments, the TKI is administered before the first administration of a recombinant viral vector. In some embodiments, the TKI is administered after the first administration of a recombinant viral vector. In some embodiments, the TKI is administered after the first administration of a recombinant viral vector and before the second administration of a recombinant viral vector. When the administration of the TKI is before or after the (first) administration of the recombinant viral vector, the administration of such 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.

[0043] In some embodiments, the recombinant viral vector is administered once or repeatedly. During the course of treating an individual with a recombinant viral vector, the recombinant viral vector can be administered once or several times. In some embodiments, the recombinant viral vector is administered once and twice.

[0044] In certain embodiments, (recombinant) viral vectors that can be used in the present invention include, but are not limited to, AAV particles. In certain embodiments, viral vectors that can be used in the present invention include, but are not limited to, retroviruses, adenoviruses, helper-dependent adenoviruses, hybrid adenoviruses, herpes simplex viruses, lentiviruses, poxviruses, Epstein-Barr viruses, vaccinia viruses, and human cytomegalovirus vectors (including recombinant versions thereof). In preferred embodiments, the recombinant viral vector comprises a lentiviral vector, an adenoviral vector, or an adeno-associated (AAV) vector.

[0045] The term "recombinant" as a modifier of viral vectors, such as recombinant AAV (rAAV) vectors, and sequences, such as recombinant polynucleotides and polypeptides, generally means that the composition has been manipulated (i.e., genetically engineered) in a manner that does not occur in nature. A specific example of a recombinant AAV vector would be when a nucleic acid (heterologous polynucleotide) not normally present in the wild-type AAV genome is inserted into the viral genome. An example would be when a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence is cloned into a vector, regardless of the presence or absence of the 5', 3', and / or intron regions with which the gene is normally associated in the AAV genome. Although the term "recombinant" is not always used herein with respect to sequences, such as AAV vectors and polynucleotides, recombinant forms containing AAV vectors, polynucleotides, and the like, are expressly included despite such omission.

[0046] A "rAAV vector" is derived from the wild-type genome of AAV, for example, by using molecular methods to remove all or part of the wild-type AAV genome and replacing it with a non-natural (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, for rAAV vectors, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained. rAAV is distinguished from the AAV genome because all or part of the AAV genome is replaced with a non-natural sequence, for example, a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence, relative to the AAV genome nucleic acid. Therefore, the integration of a non-natural (heterologous) sequence defines AAV as a "recombinant" AAV vector, which can be referred to as a "rAAV vector."

[0047] Recombinant AAV vector sequences can be packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo, and can be referred to herein as "particles." When recombinant vector sequences are encapsidated or packaged into AAV particles, the particles can also be referred to as "rAAV," "rAAV particles," and / or "rAAV virions." Such rAAV, rAAV particles, and rAAV virions contain proteins that encapsidate or package the vector genome. A specific example, in the case of AAV, is the capsid protein.

[0048] "Vector genome," sometimes abbreviated as "vg," refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form rAAV particles. When a recombinant plasmid is used to construct or produce a recombinant AAV vector, the AAV vector genome does not include portions of the "plasmid" that do not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone," and is important for plasmid cloning and amplification, processes necessary for propagation and recombinant AAV vector production, but is not itself packaged or encapsidated into rAAV particles. Thus, "vector genome" refers to the nucleic acid that is packaged or encapsidated by rAAV.

[0049] As used herein, the term "serotype" in reference to an AAV vector refers to a capsid that is serologically distinct from other AAV serotypes. Serological specificity is determined based on the lack of cross-reactivity between antibodies against one AAV compared to another AAV. Differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). Antibodies against one AAV may cross-react with one or more other AAV serotypes due to homology in the capsid protein sequences.

[0050] Under the traditional definition, a serotype means that the virus of interest has been tested against all existing and characterized serotype-specific sera for neutralizing activity, and no antibodies that neutralize the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, there may or may not be serological differences from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, it may be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are separate serotypes according to the traditional definition of serotype. Therefore, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and serologically non-distinguishable viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0051] rAAV virus vectors include any virus strain or serotype.For example, but not limited to, rAAV vector genome or particle (capsid such as VP1, VP2, and / or VP3) can be based on any AAV serotype, such as 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 can be different from each other.For example, but not limited to, rAAV plasmid or vector genome or particle (capsid) based on one serotype genome can be identical in one or more capsid proteins that package the vector. Furthermore, rAAV plasmids or vector genomes can be based on AAV serotype genomes that differ from one or more capsid proteins that package the vector genome, where at least one of the three capsid proteins can be a different AAV serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8, or a variant thereof. More specifically, a rAAV2 vector genome can contain AAV2 ITRs but can include a capsid from a different serotype, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8, or a variant thereof. Thus, rAAV vectors contain gene / protein sequences identical to those characteristic of particular serotypes, as well as "mixed" serotypes, which may also be referred to as "pseudotypes."

[0052] In certain embodiments, the rAAV plasmid or vector genome or particle is based on a reptilian or invertebrate AAV variant, such as a snake and lizard parvovirus (Penzes et al., 2015, J. Gen. Virol., 96:2769-2779) or an insect and shrimp parvovirus (Roekring et al., 2002, Virus Res., 87:79-87).

[0053] In certain embodiments, the recombinant plasmid or vector genome or particle is based on a bocavirus variant. Human bocavirus variants are described, for example, in Guido et al., 2016, World J. Gastroenterol., 22:8684-8697.

[0054] 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.

[0055] In one embodiment, the recombinant AAV (rAAV) vector comprises a VP1, VP2, and / or VP3 capsid protein having 70% or greater sequence identity to 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. In one embodiment, the recombinant AAV (rAAV) vector comprises a VP1, VP2, and / or VP3 capsid protein that has 100% sequence identity to 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. 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, ITRs.

[0056] 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 thereof (e.g., ITR and capsid variants, e.g., amino acid insertions, additions, substitutions, and deletions), as described, for example, in WO 2013 / 158879 (International Publication No. US2013 / 037170), WO 2015 / 013313 (International Publication No. US2014 / 047670), and U.S. Patent Application Publication No. 2013 / 0059732 (U.S. Patent Application No. 13 / 594,773).

[0057] 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 that have 70% or more sequence identity to 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 that have 100% or more sequence identity to VP1, VP2, and / or VP3 capsid proteins selected from the group consisting of AAV2, AAV8, and AAV8 VP1, VP2, and / or VP3 capsid proteins.

[0058] 24. The recombinant viral vector, TKI, use, or method of claim 19, 21, or 22, wherein the AAV vector comprises VP1, VP2, and / or VP3 capsid proteins that have 100% sequence identity to VP1, VP2, and / or VP3 capsid proteins selected from the group consisting of: VP1, VP2, and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8.

[0059] rAAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, and variants, hybrids, and chimeric sequences can be constructed using recombinant techniques known to those skilled in the art, so as to contain 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, as required for the rescue, replication, and packaging of recombinant vectors into rAAV vector particles.Therefore, the rAAV vector genome will contain the sequences (such as functional ITR sequences) required in cis for replication and packaging.

[0060] In certain embodiments, the lentivirus used in the present invention may be human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 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 into non-dividing cells both in vitro and in vivo. Various 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 Nov;72(ll):8463-71, 1998), U.S. Pat. Nos. 6,013,516 and 5,994,136 (any of which may be suitable viral vectors for use in the present invention).

[0061] An immune response, such as humoral immunity, can also be generated against the recombinant viral vector and / or the heterologous polynucleotide encapsidated by the viral vector or the protein or peptide encoded by the heterologous polynucleotide, 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 a subject to which the viral vector is administered.

[0062] Antibodies (such as ADA) that bind to viral vectors used in the present invention, such as recombinant viral vectors, which may be referred to as "neutralizing" antibodies, can reduce or inhibit cell transduction by viral vectors useful for gene therapy. As a result, without being bound by theory, cell transduction is reduced or inhibited, thereby reducing the introduction of heterologous polynucleotides packaged in the virus into cells and their subsequent expression, and, if necessary, their subsequent translation into proteins or peptides. Furthermore, antibodies that bind to heterologous polynucleotides encapsidated by viral vectors or proteins or peptides encoded by heterologous polynucleotides can inhibit the expression of the heterologous polynucleotide, the function or activity of the heterologous polynucleotide, or the function or activity of the protein or peptide encoded by the heterologous polynucleotide.

[0063] Thus, there may be antibodies (such as ADA) that bind to the recombinant viral vector (e.g., AAV) and / or there may be antibodies that bind to the protein or peptide encoded by the heterologous polynucleotide in the subject. Additionally, there may be antibodies that bind to the heterologous polynucleotide encapsidated by the recombinant viral vector.

[0064] Antibodies that bind to a recombinant viral vector (e.g., AAV) or to a protein or peptide encoded by a heterologous polynucleotide, when induced, can be reduced or abolished in a subject by the use of a tyrosine kinase inhibitor (TKI) as described herein.

[0065] In one embodiment, the ADA comprises IgG, IgM, IgA, IgD, and / or IgE. IgG, IgM, IgA, IgD, and / or IgE antibodies that bind to a recombinant viral vector (e.g., rAAV) or to a protein or polypeptide encoded by a heterologous polynucleotide, when induced, can be reduced or abolished in a subject by using a tyrosine kinase inhibitor (TKI) as described herein. 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, the ADA comprises IgG and / or IgG.

[0066] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotide, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).Nucleic acid includes genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA, and inhibitory DNA or RNA (e.g., RNAi, such as small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA).

[0067] 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.

[0068] A "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 the polynucleotide into a cell. A heterologous nucleic acid sequence is distinct from, i.e., non-native with respect to, the viral nucleic acid. Upon transfer / delivery into a cell, a heterologous nucleic acid sequence contained within a vector may be expressed (e.g., transcribed and, if necessary, translated). Alternatively, a transferred / delivered heterologous polynucleotide in a cell contained within a vector need not be expressed. Although the term "heterologous" is not always used herein with respect to nucleic acid sequences and polynucleotides, reference to a nucleic acid sequence or polynucleotide without the modifier "heterologous" is intended to include heterologous nucleic acid sequences and polynucleotides despite the omission.

[0069] "Transgene" is used herein to refer to a nucleic acid that is intended or introduced into a cell or organism. A transgene includes any nucleic acid, such as a heterologous polynucleotide sequence or a heterologous nucleic acid that encodes a protein or peptide. The terms transgene and heterologous nucleic acid / polynucleotide sequence are used interchangeably herein.

[0070] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and may be performed prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, remission or palliation of disease symptoms, and recovery or improved prognosis.

[0071] In one embodiment, the individual has a lung disease (e.g., cystic fibrosis), a bleeding disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), 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 type II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type I (sialidosis types I and II), type II (I-cell disease), type III (pseudo-Hurler disease) and types IV, mucopolysaccharidoses (Hurler disease and variants, Hunter disease, Sanfilippo disease types A, B, C, and D, Morquio disease types A and B, Maroteaux-Lamy disease, and Sly disease), Niemann-Pick disease types A / B, C1 and C2, and Schindler disease types I and II), hereditary angioedema (HAE), copper or iron storage disorders (e.g., Wilson disease or Menkes disease), lysosomal acid lipase deficiency, neuropathic or neurodegenerative disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic disorders (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.

[0072] In one embodiment, the individual has a blood clotting disorder, ie, hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, a deficiency of any of the following clotting factors: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma carboxylase deficiency.

[0073] In one embodiment, the individual has anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors), or a platelet disorder, e.g., Bernard-Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

[0074] In one embodiment, the individual has 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).

[0075] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of GAA (acid alpha-glucosidase) for the treatment of Pompe disease; ATP7B (copper-transporting ATPase 2) for the treatment of Wilson's disease; alpha-galactosidase for the treatment of Fabry's disease; ASS1 (argininosuccinate synthase) for the treatment of citrullinemia type 1; beta-glucocerebrosidase for the treatment of Gaucher disease type 1; beta-hexosaminidase A for the treatment of Tay-Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for the treatment of hereditary angioedema (HAE), also known as C1 inhibitor deficiency types I and II; and glucose-6-phosphatase for the treatment of glycogen storage disease type I (GSDI).

[0076] In certain embodiments, the heterologous polynucleotide is 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), angiopoietin, 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 (TGFa), and the like. ), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, 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, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0077] In certain embodiments, the heterologous polynucleotide encodes acid alpha-glucosidase (GAA). Administering a recombinant viral vector containing a heterologous polynucleotide encoding GAA to a subject with Pompe disease or another glycogen storage disease can result in the expression of GAA protein. Expression of GAA protein in a patient can suppress, inhibit, or reduce glycogen accumulation, potentially helping to prevent glycogen accumulation or decompose glycogen, thereby reducing or diminishing one or more adverse effects of Pompe disease or another glycogen storage disease.

[0078] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of thrombopoietin (TPO), interleukins (such as IL-1 through IL-36), monocyte chemotactic proteins, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors a and b, interferons 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.

[0079] In certain embodiments, the heterologous polynucleotide is selected from the group consisting of CFTR (cystic fibrosis transmembrane conductance regulator protein), blood clotting (clotting) factors (such as factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, and protein C), gain-of-function blood clotting factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metalloproteinases, and the like. transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, beta-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, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor a and beta, cytokines Interferon, α-interferon, β-interferon, interferon-g, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (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 (choroideremia), LCA5 (LCA-reversillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR type of retinitis pigmentosa: RP),Encoding DFNB1 (connexin 26 deafness), ACHM2, 3, 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, or one or more donor sequences used as repair templates for genome editing.

[0080] In certain embodiments, the heterologous polynucleotide is selected from the group consisting of erythropoietin (EPO) for the treatment of anemia; interferon-alpha, interferon-beta, and interferon-gamma for the treatment of various immune disorders, viral infections, and cancer; interleukins (ILs), including any one of IL-1 through IL-36 and their corresponding receptors, for the treatment of various inflammatory diseases or immune deficiencies; chemokines, including chemokine (C-X-C motif) ligand 5 (CXCL5), for the treatment of immune disorders; granulocyte colony-stimulating factor (G-CSF) for the treatment of immune disorders such as Crohn's disease; granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment of various human inflammatory diseases; macrophage colony-stimulating factor (M-CSF) for the treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for the treatment of epithelial tissue damage; chemokines, such as monocyte chemoattractant protein-1 (MCP-1), for the treatment of recurrent miscarriage, HIV-related complications, and insulin resistance; tumor necrosis factor (TNF) and receptors for the treatment of various immune disorders; alpha-antitrypsin for the treatment of emphysema or chronic obstructive pulmonary disease (COPD); mucopolysaccharidosis type 1 (MPS ornithine transcarbamylase (OTC) for the treatment of OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia-lyase (PAL) for the treatment of phenylketonuria (PKU); lipoprotein lipase for the treatment of lipoprotein lipase deficiency; apolipoprotein (Apo) AI for the treatment of apolipoprotein (Apo) AI deficiency; low-density lipoprotein for the treatment of familial hypercholesterolemia (FH). Receptor (LDL-R); albumin for the treatment of hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate synthetase; argininosuccinate lyase; arginase; fumarylacetoacetate hydrolase; porphobilinogen deaminase; cystathionine beta-synthase for the treatment of homocystinuria; branched-chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl-CoA carboxylase;Encoding methylmalonyl-CoA mutase; glutaryl-CoA dehydrogenase; insulin; pyruvate carboxylase; hepatic phosphorylase; phosphorylase kinase; glycine decarboxylase; H protein; T protein; cystic fibrosis transmembrane conductance regulator (CFTR); ATP-binding cassette, subfamily A (ABC1), member 4 (ABCA4) for the treatment of Stargardt disease; or dystrophin.

[0081] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein. A "polypeptide," "protein," and "peptide" encoded by a "polynucleotide sequence" includes, as with naturally occurring proteins, the full-length native sequence, and functional subsequences, modified forms, or sequence variants, so long as the subsequence, modified form, or variant retains some functionality of the native full-length protein. In the present invention, such polypeptides, proteins, and peptides encoded by polynucleotide sequences may, but are not necessarily, identical to endogenous proteins that are missing, or whose expression is insufficient or defective in the treated mammal.

[0082] In certain embodiments, the heterologous polynucleotide encodes an inhibitory nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, an RNAi, a ribozyme, and an shRNA.

[0083] In certain embodiments, the inhibitory nucleic acid is selected from the group consisting of the huntingtin (HTT) gene, genes associated with dentatorubral-pallidoluysian atrophy (atrophin 1, ATN1), androgen receptor on the X chromosome in spinal-bulbar muscular atrophy, human ataxin-1, -2, -3 and -7, Cav2.1 P / Q voltage-gated calcium channel (CACNA1A), TATA binding protein, ataxin 8 inverse chain (ATXN8OS), serine / threonine-protein phosphatase 2A in spinocerebellar degeneration (types 1, 2, 3, 6, 7, 8, 12, 17). a gene, a transcript of a gene, or a transcript of a gene associated with a polynucleotide repeat disease selected from the group consisting of 55 kDa regulatory subunit B beta isoform, 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) in fragile XE mental retardation, or AF4 / FMR2 family member 2; myotonin protein kinase (MT-PK) in myotonic dystrophy; frataxin in Friedreich's ataxia; a mutant of the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; a gene 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; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection; liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or graft function delayed kidney transplant or kidney failure acute renal failure; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta type 9 (PSMB9), metastatic melanoma;LMP7, also known as proteasome subunit beta type 8 (PSMB8), metastatic melanoma; MECL1, also known as proteasome subunit beta type 10 (PSMB10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein, apoptosis inhibitor B-cell CLL / lymphoma (BCL-2) in solid tumors; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase in liver tumors beta-catenin in familial adenomatous polyposis; beta-2 adrenergic receptor; glaucoma; RTP801 / Redd1, 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; and keratin 6A in pachyonychia congenita. N17K mutant proteins; influenza A virus genome / gene sequences in influenza infections; SARS coronavirus genome / gene sequences in severe acute respiratory syndrome (SARS) infections; respiratory syncytial virus genome / gene sequences in respiratory syncytial virus infections; Ebola virus genome / gene sequences in Ebola infections; hepatitis B and C virus genome / gene sequences in hepatitis B and C infections; herpes simplex virus (HSV) genome / gene sequences in HSV infections, and coxsackievirus B3 genome / gene sequences in coxsackievirus B3 infections; silencing of pathogenic alleles (allele-specific silencing) of genes such as torsin A (TORI A), pan-class I, and graft-specific HLA alleles in primary dystonia; and binding to mutant rhodopsin (RHO) genes in autosomal dominant retinitis pigmentosa (adRP).

[0084] In one embodiment, the protein encoded by heterologous polynucleotide comprises a gene-editing nuclease.In one embodiment, the gene-editing nuclease comprises zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN).In one embodiment, the gene-editing nuclease comprises functional type II CRISPR-Cas9.

[0085] For use in the present invention, recombinant viral vectors will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the recombinant viral vector, the method of administration, the scheduling of administration, and other factors known to medical professionals.

[0086] An effective amount of recombinant viral vector can be administered to prevent or treat disease. The appropriate administration route and dosage of recombinant viral vector can be determined based on the type of disease to be treated, the type of recombinant viral vector, the severity and course of the disease, the individual's clinical symptoms, the individual's clinical history and response to treatment, and the judgment of the attending physician. Dosage can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0087] The recombinant viral vector can be administered at any suitable dose. Generally, the dose is at least 1 x 10 per kilogram of subject body weight to achieve a therapeutic effect. 8 , or higher, e.g., 1×10 9 , 1×10 10 , 1×10, 1×10 12 , 1×10 13 Or 1 x 10 14 or more vector genomes (vg / kg).10 ~1×10 11 vg / kg, and 1 × 10 in dogs 12 ~1×10 13 AAV doses in the range of 1 x 10 vg / kg were effective. More specifically, doses of approximately 1 x 10 11 vg / kg ~ approx. 5×10 14 vg / kg (inclusive), or approximately 5 × 10 11 vg / kg ~ approx. 1×10 14 vg / kg (inclusive), or approximately 5 × 10 11 vg / kg ~ approx. 5×10 13 vg / kg (inclusive), or approximately 5 × 10 11 vg / kg ~ approx. 1×10 13 vg / kg (inclusive), or approximately 5 × 10 11 vg / kg or approximately 5 × 10 12 vg / kg (inclusive), or approximately 5 × 10 11 vg / kg ~ approx. 1×10 12 The dose is, for example, about 5×10 14 vg / kg, or approximately 5 × 10 14 less than about 2 × 10 vg / kg, e.g., 11 ~about 2×10 14 vg / kg (inclusive), particularly, for example, about 2×10 12 vg / kg, approximately 6×10 12 vg / kg, or approximately 2 × 10 13 The dose may be in vg / kg.

[0088] Dosages may vary and depend on the type, onset, progression, severity, frequency, duration or probability of the disease being treated, the desired clinical endpoint, previous or concurrent treatment, the subject's general health, age, sex, race or immunological competence, and other factors understood by those skilled in the art. The amount, number, frequency or duration of the dose may be increased or decreased proportionally as indicated by any adverse side effects, complications or other risk factors of treatment or therapy and the subject's condition. Those skilled in the art will understand the factors that may affect the dosage and timing required to provide an amount sufficient to provide therapeutic or prophylactic benefit.

[0089] The dose to achieve a therapeutic effect, e.g., a dose in vector genome per kilogram of body weight (vg / kg), will vary based on several factors, including, but not limited to, the route of administration, the level of heterologous polynucleotide expression necessary to achieve a therapeutic effect, the particular 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. One of skill in the art will be able to determine a recombinant viral vector genome dose range for treating a patient with a particular disease or disorder based on the above and other factors.

[0090] An "effective amount" or "sufficient amount" refers to an amount, in single or multiple doses, alone or in combination with one or more other compositions, treatments, protocols, or therapeutic regimen agents, that provides a detectable response of any duration (long-term or short-term), to any measurable or detectable extent, or for any duration (e.g., minutes, hours, days, months, years, or cure) to an expected or desired outcome or benefit in a subject. An "effective amount" or "sufficient amount" for treatment (e.g., to ameliorate or provide a therapeutic benefit or improvement) is typically effective to provide a measurable response to one, more, or all adverse symptoms, consequences, or complications of a disease, such as one or more adverse symptoms, disorders, illnesses, conditions, or complications caused by or associated with a disease, although reducing, reducing, inhibiting, suppressing, limiting, or controlling the progression or worsening of the disease is a satisfactory outcome.

[0091] The recombinant viral vector and the TKI can be administered by any suitable route, and may be administered by the same or different routes. In some embodiments, the recombinant viral vector is administered parenterally, particularly intravenously.

[0092] In some embodiments, 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 the course of treatment of the individual with the recombinant viral vector.

[0093] An effective or sufficient amount can be, but need not be, provided in a single administration, and may require multiple administrations, and may be administered alone or in combination with another composition (e.g., drug), treatment, protocol, or therapeutic regimen, but need not be. For example, the amount may be proportionally increased as indicated by the needs of the subject, the type, condition, and severity of the disease being treated, or the side effects (if any) of the treatment. Furthermore, an effective or sufficient amount, when given in a single dose or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol, or therapeutic regimen, need not be effective or sufficient, as additional doses, amounts, or durations beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols, or therapeutic regimens may be included to be considered effective or sufficient in a given subject. Amounts considered effective also include amounts that result in a reduction in the use of another treatment, therapeutic regimen, or protocol, such as the administration of recombinant GAA for the treatment of a lysosomal storage disease (e.g., Pompe disease), or the administration of recombinant coagulation factor proteins (e.g., FVIII or FIX) for the treatment of a coagulation disorder (e.g., hemophilia A (HemA) or hemophilia B (HemB)).

[0094] In the case of Pompe disease, an effective amount would be, for example, an amount of GAA that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen breakdown or removal, reduces lysosomal changes in the subject's body tissues, or improves muscle tone and / or strength and / or respiratory function in the subject. An effective amount can be determined, for example, by determining the kinetics of GAA uptake by myoblasts from plasma. A myoblast GAA uptake rate (K uptake) of approximately 141-147 nM may be considered effective (see, e.g., Maga et al., J. Biol. Chem. 2012). In animal models, plasma GAA activity levels greater than about 1,000 nmol / hr / mL, e.g., about 1,000 to about 2,000 nmol / hr / mL, have been observed to be therapeutically effective.

[0095] For HemA and HemB, generally speaking, to achieve therapeutic efficacy, blood coagulation factor concentrations greater than 1% of those found in normal individuals are believed to be required to convert a severe disease phenotype to a moderate one. The severe phenotype is characterized by joint damage and life-threatening bleeding. Blood coagulation factor concentrations greater than 5% of normal are believed to be required to convert a moderate disease phenotype to a mild disease phenotype.

[0096] FVIII and FIX levels in normal humans are approximately 150-200 ng / mL of plasma, but may be lower (e.g., in the range of approximately 100-150 ng / mL) or higher (e.g., in the range of approximately 200-300 ng / mL) and still be considered normal due to functional coagulation as determined, for example, by an activated partial thromboplastin time (aPTT) one-stage clotting assay. Thus, a therapeutic effect can be achieved in which the total amount of FVIII or FIX in a subject / human is greater than 1% of the FVIII or FIX present in a normal subject / human, e.g., 1% of 100-300 ng / mL.

[0097] The compositions may be administered to a subject as a combined composition, or may be administered separately, such as simultaneously, consecutively, or sequentially (before or after) with delivery or administration of a recombinant viral vector comprising a heterologous polynucleotide. The invention provides combinations in which the methods or uses of the invention are combined with any compound, agent, drug, therapeutic regimen, treatment protocol, process, therapy, or composition described herein or known to those of skill in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, therapy, or composition may be administered or performed before, substantially simultaneously with, or after administration of a recombinant viral vector comprising a heterologous polynucleotide to a subject.

[0098] Effective amount or sufficient amount does not need to be effective in each and every subject that is treated, nor does it need to be effective in the majority of the subjects that are treated in a given group or population.Effective amount or sufficient amount means effectiveness or sufficiency in a specific subject, not in a group or general population.As is typical for such methods, some subjects will show greater response, or show less response, or show no response to a given treatment method or use.

[0099] The term "ameliorating" refers to a detectable or measurable improvement in a subject's disease or its symptoms, or in an underlying cellular response. Detectable or measurable improvement includes subjective or objective reduction, reduction, inhibition, suppression, limitation, or control of the onset, frequency, severity, progression, or duration of a disease, or complications caused by or associated with a disease, or improvement of the symptoms or underlying causes or consequences of a disease, or reversal of a disease. In the case of Pompe, an effective amount would be, for example, an amount that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen breakdown or removal, or improves muscle tone and / or strength and / or respiratory function. In the case of HemA or HemB, an effective amount would be, for example, an amount that reduces the frequency or severity of acute bleeding episodes in a subject, or an amount that reduces clotting time, for example, as measured by a clotting assay.

[0100] Thus, pharmaceutical compositions of the present invention include compositions in which the active ingredients are contained in an amount effective to achieve the intended therapeutic purpose. Determination of a therapeutically effective dose is well within the capabilities of a skilled medical practitioner using techniques and guidance known in the art and using the teachings provided herein.

[0101] The therapeutic dose will depend on, among other factors, the subject's age and general symptoms, the severity of the abnormal phenotype, and the strength of the regulatory sequence that regulates the expression level.Therefore, the therapeutically effective amount in humans will fall within a relatively wide range that can be determined by medical professionals based on the response of individual patients to vector-based treatment.Such a dose may be administered alone or in combination with immunosuppressants or drugs.

[0102] Compositions such as pharmaceutical compositions can be delivered to subjects to allow transgene expression and optionally the production of encoded protein.In certain embodiments, pharmaceutical compositions contain sufficient genetic material to allow subjects to produce therapeutically effective amounts of blood coagulation factors, thereby improving the subject's hemostasis.In certain embodiments, pharmaceutical compositions contain sufficient heterologous polynucleotides to allow subjects to produce therapeutically effective amounts of GAA.

[0103] In certain embodiments, the therapeutic effect in a subject lasts for a period of time, e.g., 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, or 30-50 days or more, e.g., 50-75, 75-100, 100-150, 150-200 days or more. Thus, in certain embodiments, the recombinant viral vector provides a therapeutic effect.

[0104] An "individual" or "subject" herein is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, 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 embodiments, the individual or subject is a human. In some embodiments, the individual has a disease, particularly a disease that can be or should be treated by a recombinant viral vector.

[0105] In some embodiments, the individual has elevated serum levels of one or more cytokines. In some embodiments, the elevated serum levels are associated with administration of a recombinant viral vector to the individual. The elevated serum levels are particularly compared to serum levels in healthy individuals and / or in individuals (including the same individual) who have not been administered the recombinant viral vector (i.e., in such cases, the serum levels are elevated compared to serum levels in the absence of administration of the recombinant viral vector). In some embodiments, the one or more cytokines are selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6, and IL-1β.

[0106] The cytokine according to any of the embodiments of the present invention is preferably a pro-inflammatory cytokine, particularly one or more cytokines selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6, and IL-1β. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is TNF-α. In some embodiments, the cytokine is IFN-γ. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is IL-1β.

[0107] In some embodiments, treatment with or administration of a recombinant viral vector can result in a response in an individual. In some embodiments, the response can be a complete remission. In some embodiments, the response can be a sustained response after cessation of treatment. In some embodiments, the response can be a complete remission that is sustained after cessation of treatment. In other embodiments, the response can be a partial remission. In some embodiments, the response can be a partial remission that is sustained after cessation of treatment. In some embodiments, treatment with or administration of a recombinant viral vector and a TKI can improve the response compared to treatment with or administration of a recombinant viral vector alone (i.e., without a TKI). In some embodiments, treatment or administration of a recombinant viral vector and a TKI can increase the response rate of a patient population compared to a corresponding patient population treated with a recombinant viral vector alone (i.e., without a TKI).

[0108] In one aspect, the individual is at risk of developing ADA that binds to recombinant viral vector.In one embodiment, the ADA that binds to recombinant viral vector is not present in the individual before and / or after administration of TKI.Methods for measuring ADA before and / or after administration of recombinant viral vector are known in the art and are also described herein.

[0109] In one embodiment, ADA binding to the recombinant viral vector is reduced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to natural history data of a relevant control group, optionally without administration of a tyrosine kinase inhibitor (TKI). In one embodiment, the reduction in ADA is measured in the serum of an individual. The reduction in ADA serum level is particularly when compared to the serum level of an individual (including the same individual) not administered a TKI (i.e., in such cases, the serum level is reduced compared to the serum level without / before administration of the TKI). The reduction in serum level is particularly when compared to the serum level or cytokine secretion of an individual (including the same individual) administered a recombinant viral vector (particularly the first administration) but not administered a TKI (i.e., in such cases, the serum level is reduced compared to the serum level with / after administration of the recombinant viral vector but without / before administration of the TKI). Without the reduction, the serum level (of ADA) may be elevated / increased in association with (administration of) the recombinant viral vector. In some embodiments, the reduction is clinically meaningful and / or statistically significant. The reduction can be partial or complete. In some embodiments, the reduction is clinically meaningful and / or statistically significant.

[0110] In one embodiment, the individual is at risk of developing ADA to the polypeptide encoded by the heterologous polynucleotide. ADA to the heterologous polypeptide can reduce the effectiveness of the treatment, for example, by reducing the number of cells that express the heterologous polypeptide (i.e., the transgene).

[0111] In one embodiment, transgene expression increases upon readministration of the viral vector, particularly compared to transgene expression before readministration of the viral vector. In one embodiment, transgene expression increases by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to transgene expression before readministration of the viral vector. In one embodiment, transgene expression is maintained upon readministration of the viral vector, particularly compared to transgene expression before readministration of the viral vector. In one embodiment, the increase in transgene expression is measured in the serum and / or (target) tissues of the individual. The increase in transgene expression is particularly when compared to transgene expression in an individual (including the same individual) who has not been administered a TKI (i.e., in such cases, transgene expression is reduced compared to transgene expression without / before administration of the TKI). Said increase in transgene expression is particularly when compared to transgene expression in an individual (including the same individual) who has been administered a recombinant viral vector (particularly the first administration) but has not been administered a TKI (i.e., in such a case, transgene expression is increased compared to transgene expression with / after administration of the recombinant viral vector but without / before administration of the TKI). Without said increase, transgene expression may be decreased / reduced in association with (administration of) the recombinant viral vector. In some embodiments, said increase is clinically meaningful and / or statistically significant. Said increase may be partial or complete. In some embodiments, said increase is clinically meaningful and / or statistically significant.

[0112] array TIFF2025526697000002.tif54164TIFF2025526697000003.tif243164TIFF2025526697000004.tif141164 [Brief explanation of the drawings]

[0113] [Figure 1]rAAV8 rechallenge and dasatinib treatment in mice. C57Bl / 6 mice were intravenously administered rAAV8 encoding hSEAP (human secreted alkaline phosphatase) at a dose of 1E12 vg / kg on study day 1. On study day 22, mice received a second rAAV8 encoding hFIX (human factor IX) at a dose of 5E13 vg / kg. Group 1 (non-immunized mice, n = 9) did not receive the first rAAV8 injection. Group 2 (immunized mice, n = 10) received both rAAV8 injections. Group 3 mice (n = 10) received two rAAV8 injections and were treated with 50 mg / kg dasatinib by oral gavage twice daily for 7 days, starting from the day of the first rAAV8 administration. Blood samples were collected 1 day before and every 7 days after the first rAAV8 treatment. [Figure 2A] 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). Anti-AAV8 IgM was measured by ELISA against immobilized empty AAV8 capsids. IgM antibody titers were determined by ELISA OD values of serial dilutions (1:10 and 7 serial dilutions of 1:3). Readings from all mouse groups were pooled on day 0 to determine the average background level. The positivity threshold is represented by a dotted line. The kinetics of anti-AAV8 IgM antibody titers are shown for each mouse group (Figures 2A-C). Open circles represent titers below the threshold, and crossed circles represent titers above the threshold. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A]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). Anti-AAV8 IgG was measured by ELISA against immobilized empty AAV8 capsids. IgG antibody titers were determined by ELISA OD values of serial dilutions (1:10 and 7 serial dilutions of 1:3). Readings from all mouse groups were pooled on day 0 to determine the average background level. The positivity threshold is represented by a dotted line. The kinetics of anti-AAV8 IgG antibody titers are shown for each mouse group (Figures 3A-C). Open circles represent titers below the threshold, and crossed circles represent titers above the threshold. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4A] Figure 4. Dasatinib treatment enables transgene expression after rAAV rechallenge. Serum was isolated from blood drawn on study days 0, 8, 15, 21, 29, and 36 (Figure 1). (Figure 4A) Expression of the first transgene, hSEAP, was measured by chemiluminescence. (Figure 4B) After AAV8 rechallenge, expression of the second transgene, hFactorIX, was measured by ELISA. Mean concentrations are shown + / - SD. [Figure 4B] See legend to Figure 4A. [Figure 5A] Figure 5. Effect of dasatinib on AAV-mediated cytokine release in human whole blood. Low-level IFN-γ and IL-6 production in whole blood was stimulated after 24 hours of incubation with 5E11 vg / mL AAV8-hSEAP. In the presence of 12.5 or 50 nM dasatinib, this production was dose-dependently reduced. Cytokine concentrations (pg / mL + / - SD, left), fold increase over levels measured in the PBS control (center), and percentage inhibition of AAV-dependent cytokine release (right, calculated as 100 × (concentration of AAV8 alone - concentration of AAV8 and dasatinib) / (concentration of AAV8 alone)) are shown, respectively. (Figure 5A) Interferon-γ production. (Figure 5B) IL-6 production. [Figure 5B] See legend to Figure 5A. [Figure 6A] Figure 6: Dasatinib inhibits AAV8-dependent cytokine release in human whole blood. Fresh whole blood from four healthy donors was incubated for 24 hours with either PBS or AAV8-hSEAP (5e11 vg / mL) containing IVIG (PRIVIGEN, 1 / 100). 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 was added 9 hours after AAV8 treatment (dasa 50 nM 2x). After 24 hours, plasma was collected and cytokines were measured using a Quanterix kit. The fold increase in cytokines relative to PBS for IFN-γ and TNF-α (Figure 6A), IL-6 and IL-1α (Figure 6B), and IL-2 and IL-1β (Figure 6C) is shown, and the percentage of inhibition in the presence of dasatinib (mean + / - SEM) is shown. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7] In vivo study to evaluate the effects of extended dasatinib treatment. All mouse groups except group 1 were immunized with an intravenous injection of AAV8-hSeap (1e12vg / kg) on day 1. Four groups received an intravenous injection of AAV8-FactorIX (3e13vg / kg) on day 43. Concurrent with the first AAV administration, groups 3 and 4 were treated with dasatinib for 1 week or 2 weeks, respectively. Dasatinib was administered at a dose of 50 mg / kg / gavage twice daily, starting 1 hour before the first AAV treatment. [Figure 8A] Figure 8: Extending dasatinib treatment from 1 week to 2 weeks ameliorates inhibition of IgM formation against AAV8. In the experiment described in Figures 8A and 8B, 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. [Figure 8B] See legend to Figure 8A. [Figure 9A]Figure 9: Extending dasatinib treatment from 1 week to 2 weeks ameliorates inhibition of IgG formation against AAV8. In the experiment described in Figures 9A and 9B, 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. [Figure 9B] See legend to Figure 9A. [Figure 10] Prolonged dasatinib treatment during the first AAV8 administration improves transgene expression after re-administration. In the experiment described in Figure 10, the level of hFIX expression (ng / mL, median + / - SD) was measured for each group starting from day 42 (1 day before administration of AAV8-hFIX) to day 64. [Figure 11] Dasatinib treatment inhibits cytokine and chemokine release by mouse splenocytes in response to AAV8. Splenocytes were isolated from C57 / B16 mice and incubated with AAV8-hSeap at an MOI of 1E5 for 24 hours in the presence or absence of dasatinib (100 nM, 50 nM, or 12.5 nM). Cytokines and chemokines were measured in culture supernatants. LPS (positive) and PBS (negative) controls are shown on the graph. Mean + / - SD (triplicates). Dashed line: lower limit of quantitation. [Figure 12A] 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 the AAV2 and AAV9 capsid sequences in the absence (black bars) or presence (gray bars) of 100 nM dasatinib. After 48 hours, IFN-γ and TNF-α secreting cells were measured by Fluorospot. The mean spot-forming cells + / - SD are shown for 1E6 PBMCs. Dotted line: positive threshold. [Figure 12B] See legend to Figure 12A. [Example]

[0114] The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0115] Example 1. Dasatinib prevents anti-AAV8 antibody formation following rAAV8 administration in mice. To evaluate whether dasatinib can effectively prevent anti-AAV antibody formation after rAAV administration and enable efficient re-administration of rAAV, a study was conducted in mice (Figure 1). A group of C57Bl / 6 mice was intravenously administered a first rAAV8 encoding hSEAP (human secreted alkaline phosphatase under the control of the CMV promoter) at a dose of 1E12 vg / kg on study day 1. On study day 22, a group of mice was administered a second rAAV8 encoding hFIX (human factor IX under the control of the CMV promoter) at a dose of 5E13 vg / kg. The non-immunized control group 1 did not receive the first rAAV8 injection, while the immunized control group 2 received two injections. A third group of mice was treated with dasatinib at a dose of 50 mg / kg by oral gavage twice daily for 7 days, starting from the day of the first rAAV8 administration. Blood samples were collected on study day 0 (1 day before the first AAV administration), days 8, 15, 21, 29, and 36. Serum IgM and IgG against the AAV8 capsid were titrated in the serum samples (Figures 2 and 3). Anti-AAV8 IgM peaked 1 week after the first rAAV8 administration, on day 8 for the immunized control group 2 and on day 29 for the non-immunized control group 1 (10 / 10 mice above the threshold) (Figure 2). In the dasatinib-treated group 3, sera from all mice remained below the IgM positivity threshold until the second rAAV8 administration. The IgM positivity of mouse 302 on day 15 only was attributed to technical issues with the ELISA. Anti-AAV8 IgG titers were dramatically reduced in the dasatinib-treated group 3 compared with the immunized control group 2. On day 21 of the study, only 4 / 10 mice in Group 3 were above the positivity threshold, whereas 10 / 10 mice in Group 2 were above this threshold (Figure 3). On day 36, two weeks after rAAV8 rechallenge, 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 demonstrate that dasatinib effectively reduces anti-AAV8 antibody formation after rAAV8 administration in mice.

[0116] Example 2. Transient dasatinib treatment allows transgene expression after rAAV8 re-administration. Expression of the first transgene, hSEAP, and the second transgene, hFIX, was measured in serum samples from the experiment presented above (Figure 1, Example 1). As expected, human SEAP was detected in the serum of mice in Groups 2 and 3 already 7 days after AAV8-hSEAP injection on Study Day 1 (Figure 4A). After the second administration of rAAV8 on Study Day 22, hFIX transgene expression was undetectable in the serum of mice in the immunized control Group 2 (Figure 4B) and remained at baseline until Day 36 (14 days after AAV8-hFIX administration), indicating that re-administration was entirely ineffective. In contrast, hFIX expression was detected in the serum of all mice in the non-immunized Group 1 (mean: 2107 ng / ml) and most mice in the dasatinib-treated Group 3 (mean: 1398 ng / ml). This data indicates that dasatinib treatment during the first rAAV administration at least partially restored the efficacy of transduction by the second rAAV. Overall, the results from Examples 1 and 2 demonstrate that inhibition of anti-AAV8 antibody formation resulting from transient dasatinib treatment allows for efficient AAV re-administration.

[0117] Example 3. Dasatinib dose-dependently inhibits AAV-induced cytokine production in human blood. To assess whether dasatinib could affect AAV-mediated cytokine release, we performed an assay on whole blood from a healthy donor. This donor was seropositive for anti-AAV8 antibodies (IgG titer: 1 / 21870; IgM titer: 1 / 810). Whole blood from an AAV8 pre-immune donor 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 robust 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 to assess baseline cytokine production. Dasatinib was added to blood 1 hour before incubation with AAV8-hSEAP. After 24 hours, plasma supernatants were collected, and cytokines were measured using a Quanterix kit with an SP-X Imaging and Analysis System (Simoa). Figures 5A and 5B show that low levels of IFN-γ and IL-6 production in whole blood were stimulated after 24 hours of incubation with 5E11 vg / mL AAV8-hSEAP. In the presence of 12.5 or 50 nM dasatinib, this production was reduced in a dose-dependent manner. These results suggest that inhibition of cytokine release may be the mechanism by which dasatinib inhibits anti-AAV antibody production.

[0118] Example 4. Dasatinib dose-dependently inhibits AAV-induced cytokine production in human blood. 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 (5e11 vg / ml) for 24 hours. Intravenous immunoglobulin (IVIG, Privigen) containing anti-AAV antibodies was added to the blood samples. Dasatinib was added at 50 nM 1 hour before AAV treatment, and PBS was used as a negative control. Because dasatinib has a very short half-life (Lindauer M et al., Recent Results Cancer Res. 2010;184:83-102), a second dasatinib addition was performed 9 hours after AAV treatment. After 24 hours, plasma supernatants were collected, and cytokines were measured using a Quanterix kit. Figure 6 shows that the production of IFN-γ, IL-6, IL-2, TNF-α, IL-1α, and IL-1β was inhibited in the presence of dasatinib. This inhibition was more pronounced when dasatinib was added both 1 hour before and 9 hours after AAV treatment. These results indicate that dasatinib inhibits the release of several pro-inflammatory 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 likely contributes to dasatinib's effect on antibody formation.

[0119] Example 5. Prolonged dasatinib treatment improves anti-AAV8 antibody inhibition after rAAV8 administration and allows for efficient re-administration in mice. In the experiment described in Example 1, despite the strong inhibition of anti-AAV antibody formation observed in most mice, four of ten dasatinib-treated animals began to express anti-AAV8 IgG before rechallenge (Figure 3C, days 15–21). The presence of residual AAV8 particles in the circulation and in tissues after cessation of dasatinib treatment may lead to this antibody formation. Therefore, we investigated whether prolonged dasatinib treatment could more efficiently inhibit humoral responses to AAV8 capsids. To this end, we applied the same protocol as in Example 1, but used either 1-week or 2-week dasatinib treatment. Furthermore, rechallenge was performed at a later time point (day 43 instead of day 22) so that potential antibody formation could be observed after cessation of dasatinib treatment. This protocol is illustrated in Figure 7.

[0120] Circulating IgM and IgG against the AAV8 capsid were titrated in serum samples (Figures 8 and 9). Peak anti-AAV8 IgM was observed 1 week after the first rAAV8 administration, at day 8 for the immunized control group 2 and at day 50 for the non-immunized control group 1 (10 / 10 mice above the threshold) (Figure 8). In group 3, treated with dasatinib for 1 week, IgM titers remained low to negative until the second rAAV8 administration. At day 42, before AAV8 rechallenge, 4 of 15 mice had positive IgM titers against AAV8. Inhibition of IgM production was stronger in group 4, treated with dasatinib for 2 weeks. In this group, only one mouse showed a positive IgM titer at day 42.

[0121] In Group 3, treated with dasatinib for one week, 3 / 15 mice showed negative anti-AAV8 IgG titers on day 42, whereas all 15 mice in the immunized control Group 2 had very high IgG titers (Figure 9). Regarding IgM, the inhibition of IgG formation was stronger in Group 4 mice treated with dasatinib for two 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 in Group 3. In conclusion, extending dasatinib treatment from one week to two weeks resulted in stronger inhibition of both IgM and IgG formation against AAV8.

[0122] On day 64, 3 weeks after rAAV8 rechallenge, 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.

[0123] Inhibition of antibody formation against AAV8 correlated with improved hFIX transgene expression after rechallenge, indicating efficient transduction by the second AAV8 (Figure 10). hFIX expression was inversely correlated with IgG and IgM titers measured on day 42 before rechallenge. In mice that remained IgG-negative on day 42, hFIX levels on day 64 were within the range of those measured in the non-immunized control group 1. hFIX expression was higher in group 4, which was treated with dasatinib for 2 weeks, reflecting a more complete inhibition of anti-AAV8 antibody formation compared with group 3, which was treated for only 1 week.

[0124] Example 6: Dasatinib reduces AAV8-dependent cytokine and chemokine release in mouse splenocytes. 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 hours in the presence or absence of different concentrations of dasatinib. Cytokines and chemokines secreted into the culture supernatant were analyzed using ProcartaPlex Immunoassays (Luminex). As shown in Figure 11, a dose-dependent decrease was measured in various cytokines (IL-6, TNF-α) and chemokines (IP-10 / CXCL10, MCP-1, MCP-3, MIP-1α, MIP-1β, MIP-2α) characteristic of the early innate immune response to rAAV (Shirley JL et al. Mol Ther. 2020 Mar 4;28(3):709-722).

[0125] Together with Examples 3 and 4, which illustrate the effect of dasatinib on cytokine production by human PBMCs, these results indicate that dasatinib downregulates the innate immune response to AAV in both humans and mice. These results also suggest that the inhibition of antibody responses to AAV vectors observed in mice may be at least partially the 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).

[0126] Example 7. Dasatinib inhibits human T cell responses to AAV2 and AAV9 in vitro. The above examples demonstrate that dasatinib treatment has the potential to inhibit both innate and humoral immune responses to AAV8. A key issue has been its potential effect on the cellular adaptive immune response. It has been reported that T cell responses to rAAV induce clearance of AAV-transduced cells, thereby reducing the duration of transgene expression. It is also known that T cell responses to AAV can mediate liver toxicity in the clinic.

[0127] T cell responses to AAV can be measured in healthy human blood donors previously infected with wild-type AAV. The FluoroSpot assay reveals the percentage of IFN-γ- and TNF-α-producing PBMCs induced upon incubation with pools of AAV capsid peptides. We used this assay to evaluate the responses 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 positive IFN-γ responses to AAV9 pool 1 and TNF-α responses 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 responses were inhibited in the presence of 100 nM dasatinib.

[0128] These results indicate that dasatinib has the potential to block both T cell and antibody responses to the adaptive immune response against the AAV capsid. This suggests that this compound may effectively mitigate the immunogenicity of AAV-based gene therapy vectors. Importantly, the inhibitory effect of dasatinib is not limited to the immune response against the AAV8 serotype, as T cell responses against AAV2 and AAV9 were also inhibited by dasatinib treatment.

[0129] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the illustrations 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 entireties.

Claims

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

2. 1. Use of a recombinant viral vector comprising a heterologous polynucleotide in the manufacture of a medicament for the treatment of a disease in an individual, said treatment comprising: (a) administering the recombinant viral vector to the individual; and (b) administering to said individual a tyrosine kinase inhibitor (TKI) to prevent or reduce the formation of anti-drug antibodies (ADA) associated with said administration of said recombinant viral vector. Including, use.

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

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

5. 1. 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 containing a heterologous polynucleotide to an individual.

6. A method for preventing or alleviating the formation of anti-drug antibodies (ADA) associated with the administration of a recombinant viral vector containing 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 claims 1 to 6, wherein said TKI is a Lck and / or Src kinase inhibitor, in particular dasatinib.

8. The administration of the TKI (i) inhibiting the formation of ADA that binds to the recombinant viral vector; (ii) inhibition of T cell activation (induced by said recombinant viral vector); (iii) inhibition of T cell cytotoxic activity (induced by the recombinant viral vector); (iv) inhibition of B cell activation (induced by said recombinant viral vector); (v) inhibiting the formation of plasma cells (induced by said recombinant viral vector); and / or (vi) Inhibition of cytokine secretion by immune cells (induced by said recombinant viral vector), particularly wherein said cytokine is one or more cytokines selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6, and IL-1β. The recombinant viral vector, TKI, use, or method according to any one of claims 1 to 7, which causes

9. 9. The recombinant viral vector, TKI, use or method according to any one of claims 1 to 8, wherein (administration of) said TKI causes a reduction in serum levels of one or more cytokines in said individual, in particular said one or more cytokines 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 one of claims 1 to 9, wherein the administration of the TKI is (i) before, simultaneously with, or after administration of the recombinant viral vector, (ii) intermittently or continuously, and / or (iii) orally.

11. The administration of the TKI (i) inhibiting the formation of ADA that binds to the recombinant viral vector; (ii) inhibition of T cell activation (induced by said recombinant viral vector); (iii) inhibition of T cell cytotoxic activity (induced by the recombinant viral vector); (iv) inhibition of B cell activation (induced by said recombinant viral vector); (v) inhibiting the formation of plasma cells (induced by said recombinant viral vector); and / or (vi) Inhibition of cytokine secretion by immune cells (induced by said recombinant viral vector), in particular, wherein said cytokine is one or more cytokines selected from the group consisting of IL-2, TNF-α, IFN-γ, IL-6 and IL-1β. The recombinant viral vector, TKI, use, or method of any one of claims 1 to 10, wherein the recombinant viral vector, TKI, use, or method is administered at a dose sufficient to cause

12. The recombinant viral vector, TKI, use, or method of any one of claims 1 to 11, wherein the administration of the TKI is at a dose sufficient to cause a decrease in serum levels of one or more cytokines in the individual.

13. The recombinant viral vector, TKI, use, or method of any one of claims 1 to 12, wherein the administration of the TKI is at a dose sufficient to cause a decrease in 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 claims 1 to 13, wherein the administration of the TKI is at an effective dose.

15. 15. The recombinant viral vector, TKI, use, or method according to any one of claims 1 to 14, 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, in particular at a dose of about 100 mg or less.

16. 16. The recombinant viral vector, TKI, use, or method of any one of claims 1 to 15, wherein administration of the TKI is for a period during which ADA formation is expected and / or is stopped after prevention or reduction of ADA formation.

17. 17. The recombinant viral vector, TKI, use or method of any one of claims 1 to 16, wherein administration of the TKI is concomitant with a first administration of the recombinant viral vector, and optionally occurs before, simultaneously with or after the first administration of the recombinant viral vector.

18. said administering said recombinant viral vector (i) administration at an effective dose; (ii) parenteral, especially intravenous, administration; and / or (iii) a first administration of the recombinant viral vector to the individual; A recombinant viral vector, TKI, use or method according to any one of claims 1 to 17.

19. 19. The recombinant viral vector, TKI, use, or method of any one of claims 1 to 18, wherein the recombinant viral vector comprises a lentiviral vector, an adenoviral vector, or an adeno-associated (AAV) vector.

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

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

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

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

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

25. 25. The recombinant viral vector, TKI, use or method of any one of claims 1 to 24, wherein the individual is at risk of developing ADA associated with the recombinant viral vector.

26. 26. The recombinant viral vector, TKI, use or method of any one of claims 1 to 25, wherein ADA bound to said recombinant viral vector is not present in said individual before and / or after administration of said TKI.

27. The recombinant viral vector, TKI, use or method of any one of claims 1 to 26, wherein said individual is at risk of developing ADA to the polypeptide encoded by said heterologous polynucleotide.

28. The recombinant viral vector, TKI, use or method of any one of claims 1 to 27, wherein the ADA comprises IgG, IgM, IgA, IgD and / or IgE, in particular the ADA comprises IgG and / or IgM.

29. 29. The recombinant viral vector, TKI, use or method of any one of claims 1 to 28, wherein the ADA binding to the recombinant viral vector is reduced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to natural history data of a relevant control group, optionally without administration of the TKI.

30. 30. The recombinant viral vector, TKI, use or method of any one of claims 1 to 29, wherein transgene expression is increased upon readministration of the viral vector, particularly compared to the transgene expression before readministration of the viral vector.

31. 31. The recombinant viral vector, TKI, use or method of any one of claims 1 to 30, wherein transgene expression is increased by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the transgene expression before readministration of the viral vector.

32. 32. The recombinant viral vector, TKI, use or method of any one of claims 1 to 31, wherein transgene expression is maintained upon readministration of said viral vector, particularly compared to said transgene expression before readministration of said viral vector.

33. The invention as hereinbefore described.

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