Method and use of apheresis

Abstract

To provide a method of treating a disease caused by loss of function or activity of protein.SOLUTION: A method includes: (a) a step of removing, reducing, depleting, inhibiting, inactivating, or capturing an AAV binding antibody from a blood product obtained from the subject by a process including apheresis; and (b) a step of administering a certain amount of recombinant adeno-associated virus (rAAV) vector containing heterologous polynucleotide coding protein or peptide which provides or assists the function or activity of the protein.SELECTED DRAWING: None

Description

[Background technology]

[0001] Introduction

[0002] Gene therapy using recombinant adeno-associated virus (rAAV) (gene transfer) shows promising potential to address unmet medical needs. For example, gene therapy using AAV expressing coagulation factors VIII and IX has shown promising safety and efficacy in human clinical trials (references).

[0002]

[0003] AAV infection is common in the human population and is not known to cause disease. The majority of human subjects who can benefit from AAV-based gene therapy are those who have been previously infected with AAV. Infection can occur later in life, but most frequently in childhood. As with all viral infections, the host immune response to AAV infection results in the formation of antibodies against AAV (AAV antibodies). A well-known time series follows AAV exposure: rapid AAV antibody formation (weeks), achievement of a high titer antibody peak, and subsequent gradual decline in AAV antibody levels (years) (references). The typical peak titer of AAV antibodies after AAV infection is >1:100 and can easily exceed 1:1000 (Calcedo et al, 2009).

[0003]

[0004] In promising hemophilia clinical trials to date (George et al 2016, American Society for Hematology, San Diego, CA, Plenary Lecture; George et al 2017, International Society for Thrombosis and Hemostasis, Berlin, Germany), the best results have been observed after systemic (e.g., intravenous) administration of an AAV vector expressing a therapeutic transgene (FVIII or FIX) to human subjects without existing antibodies (titer <1:1). Good results can also be obtained with very low titers of existing antibodies (1:1 to 1:2), and moderate results may be obtained with low titers of existing antibodies (1:3 to 1:5). Existing antibody levels above these levels correspond to near the lower limit to insufficient gene transduction. The mechanism of this decrease in gene transfer efficiency as a function of existing antibody titers is the binding and neutralization of the AAV gene therapy vector by existing AAV antibodies. When anti-AAV antibodies bind to the vector, the vector cannot reach and introduce the therapeutic gene into target tissues and cells such as hepatocytes and endothelial cells, which are targets for the gene transfer. Depending on the specific AAV serotype, subjects with up to 50% and over 50% hemophilia may not be eligible to benefit from AAV-based gene therapy treatment due to pre-existing AAV antibodies (Calcedo et al 2009). [Overview of the project]

[0004]

[0005] Methods and uses for removing, depleting, capturing, and / or inactivating AAV antibodies in prospective mammals, such as human subjects, that may benefit from AAV gene therapy are disclosed herein. In certain embodiments, AAV antibodies are present in levels that reduce or block the transduction of therapeutic gene transfer vectors into target cells. In certain embodiments, AAV antibodies are pre-existing and can be present in levels that reduce or block the transduction of therapeutic gene transfer vectors into target cells. In certain embodiments, AAV antibodies may develop after exposure to AAV or administration of AAV vectors for gene therapy. If such antibodies develop after administration of AAV vectors for gene therapy, these subjects can also be treated according to the present invention.

[0005]

[0006] The method is based on a medical device / procedure generally referred to as apheresis, more particularly plasmapheresis involving blood products. In certain embodiments, apheresis is used to provide the benefits of AAV gene therapy, particularly in subjects who already have AAV antibodies or who develop AAV antibodies after gene therapy.

[0006]

[0007] Generally, apheresis or plasmapheresis is a method of circulating human subject plasma ex vivo (outside the body) through a device that modifies the plasma by adding, removing and / or exchanging components before returning it to the patient. Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from blood products (e.g., plasma). This procedure depletes, captures, inactivates, reduces or removes immunoglobulins (antibodies) that bind to AAV, thereby reducing the titer of AAV antibodies in the subject being treated, and thereby reducing AAV antibodies that can contribute to the neutralization of AAV. A device useful in carrying out the present invention may be in the form of an AAV capsid affinity matrix column. Passing a human subject blood product (e.g., plasma) through an AAV capsid affinity matrix results in the binding of AAV antibodies, and only all isotypes (including IgG, IgM, etc.).

[0007]

[0008] Sufficient plasmapheresis using an AAV capsid affinity matrix is ​​expected to substantially remove AAV capsid antibodies and reduce AAV capsid antibody titers (loads) in humans treated in this manner. In certain embodiments, the titer in the treated subject is reduced to a substantially low level (<1:5, or less, e.g., <1:4, or <1:3, or <1:2, or <1:1). The reduction in antibody titer is expected to be temporary because the B lymphocytes producing AAV capsid antibodies are expected to gradually cause the AAV capsid antibody titer to rebound to the steady-state level before the plasmapheresis procedure intervention. The kinetics of this rebound are based on the half-life of IgG (20 hours) and the fact that the synthesis rate is equal to the decay rate for the system in a steady state (corresponding to the steady-state AAV capsid titer before the plasmapheresis procedure).

[0008]

[0009] When the existing capsid antibody titer is reduced from 1:100 to 1:1, rebounds in AAV antibody titers of approximately 0.15% (corresponding to a titer of 1:1.2), 0.43% (1:1.4), 0.9% (1:1.9), 1.7% (1:2.7), and 3.4% (1:4.4) occur 1, 3, 6, 12, and 24 hours after completion of the plasmapheresis procedure, respectively. The transient removal of AAV antibodies (e.g., AAV antibodies that bind to the AAV capsid) from such subjects corresponds to a certain time frame (e.g., approximately 24 hours or less, e.g., 12 hours or less, or 6 hours or less, or 3 hours or less, or 2 hours or less, or 1 hour or less), during which the therapeutic AAV vector can be administered to the subject and efficiently transduced into the target tissue without significant neutralization of the AAV vector by AAV antibodies.

[0009]

[0010] When the existing capsid antibody titer is reduced from 1:1000 to 1:1, rebounds in AAV antibody titers of approximately 0.15% (corresponding to a titer of 1:2.5), 0.4% (1:5.3), 0.9% (1:9.7), 1.7% (1:18), and 3.4% (1:35) occur 1, 3, 6, 12, and 24 hours after completion of the plasmapheresis procedure, respectively. Therefore, the window for AAV vector administration is relatively short.

[0010]

[0011] Parameters such as the type of AAV capsid affinity matrix can be modified according to the (one or more) AAV antibody serotypes in the subject. Therefore, the AAV capsid affinity matrix can be adjusted (increased or decreased) according to the (one or more) AAV antibody serotypes in the subject. For example, if the antibody binds to one or more serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1 and SEQ ID NO: 2, then antibodies specific to one or more of the (one or more) AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1 and SEQ ID NO: 2 can be used as the affinity matrix.

[0011]

[0012] Parameters such as the amount of AAV capsid affinity matrix can be modified according to the AAV antibody titer in the subject. For example, the amount of AAV capsid affinity matrix can be adjusted (increased or decreased) according to the amount of AAV antibody in the subject. For high AAV antibody titers, the amount of AAV capsid affinity matrix can be increased. For lower AAV antibody titers, the amount of AAV capsid affinity matrix can be relatively reduced.

[0012]

[0013] Parameters such as the amount of AAV capsid affinity matrix can also be modified according to the volume of blood product being processed from the subject. For example, the amount of AAV capsid affinity matrix can be adjusted (increased or decreased) according to the volume of blood product in contact with the matrix.

[0013]

[0014] In addition, the time frame after AAV antibody depletion, capture, inactivation, or removal can be modified depending on how quickly the AAV antibody rebounds. For example, the rebound of AAV antibodies may be faster or slower in certain subjects. If the rebound of AAV antibodies is faster, the time frame in which therapeutic AAV vectors can be administered to the target will be relatively shorter. If the rebound of AAV antibodies is slower, the time frame in which therapeutic AAV vectors can be administered to the target will be relatively longer. [Modes for carrying out the invention]

[0014]

[0015] AAV vectors possess numerous desirable features for such applications, including targeting for both dividing and non-dividing cells. Early clinical experience with these vectors has demonstrated long-term expression in treated humans. In addition, early clinical trials have demonstrated the absence of persistent toxicity and minimal or undetectable immune responses. AAV is known to infect diverse cell types in vivo and in vitro via receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting numerous tissues such as retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0015]

[0016] The present invention provides compositions and methods for removing, depleting, capturing, and / or inactivating AAV-binding antibodies. Such antibodies can pre-exist in a subject, such as a mammalian subject, for example a human. Alternatively, such AAV-binding antibodies can arise in a mammalian subject, such as a human, due to exposure to AAV or treatment / administration of an AAV vector to the subject. The compositions and methods of the present invention utilize an AAV-binding antibody affinity matrix.

[0016]

[0017] In some embodiments, the AAV-binding antibodies are removed, depleted, captured, and / or inactivated from a blood preparation obtained from a subject by a method including apheresis. Non-limiting examples of apheresis include apheresis, plasma exchange, cytapheresis, or combinations thereof. Apheresis refers to a method of ex vivo manipulation, removal, depletion, and / or inactivation of components present in a subject's blood or blood preparation. In some embodiments, the blood or blood preparation is returned to the subject after apheresis.

[0017]

[0018] In a typical apheresis method, blood is obtained directly from a subject's vein or artery. In some embodiments, the blood is separated into two or more blood preparations, components (e.g., cells or proteins) are removed from one of the blood preparations, and the blood preparations are optionally combined. The blood is optionally returned directly to the subject's artery or vein.

[0018]

[0019] More specifically, for example, in an apheresis method, peripheral blood is removed from the subject by a suitable apheresis column or machine, an anticoagulant is optionally added to the blood, and the blood is separated into a cellular fraction (e.g., including erythrocytes, leukocytes, and platelets) and a liquid fraction (e.g., plasma). The liquid fraction is then subjected to apheresis, in which components in the liquid fraction (AAV-conjugated antibodies) are removed, depleted, captured, and / or inactivated. The processed plasma can then be combined with the previously separated solid blood components and reinjected into the subject. Suitable methods and apparatus for separating plasma from whole blood are known to those skilled in the art, for example, as described in U.S. Patent No. 4,619,639, and any volume loss due to apheresis can be subsequently replaced with a suitable solution such as isotonic saline.

[0019]

[0020] In certain embodiments, the apheresis method removes, depletes, captures and / or inactivates at least 20%–50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of AAV-binding antibodies from the blood product obtained from the subject. In certain embodiments, the method removes, depletes, captures and / or inactivates at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of AAV-binding antibodies from the blood product obtained from the subject. Non-limiting examples of blood products include whole blood, serum, plasma, and combinations thereof. Blood products may be cell-deficient or may contain cells (e.g., erythrocytes, platelets, and / or lymphocytes).

[0020]

[0021] Any AAV-binding antibody affinity matrix can be attached to or immobilized on the substrate for use in the preparation of compositions or in apheresis methods using any preferred method. For example, in one embodiment, an antibody that binds to an AAV-binding antibody can be attached to or immobilized on the substrate for an apheresis column or apheresis method as disclosed herein. In another embodiment, an AAV capsid protein or AAV capsid fragment can be attached to or recruited on the substrate for an apheresis column or apheresis method as disclosed herein. Such AAV capsid proteins and fragments include VP1, VP2 and / or VP3 of any AAV serotype suitable for use in the preparation of compositions or in apheresis methods.

[0021]

[0022] The affinity matrix that binds to the AAV antibody can be immobilized on a substrate using any preferred method and any preferred substrate. In some embodiments, the affinity matrix immobilized on the substrate of the affinity column is suitable for apheresis applications. In some embodiments, the apheresis process includes an apheresis method, device, or column as disclosed in U.S. Patents 9,726,666 and 8,877,177.

[0022]

[0023] The substrate on which the affinity matrix that binds to the AAV antibody is immobilized is typically a solid substrate. “Solid substrate” refers, for example, to a material having one or more rigid or semi-rigid surfaces, the surfaces of which may have a regular or irregular geometric configuration, and which can take the form of beads, resins, gels, spheres, microspheres, particles, fibers, or other geometric or physical forms. Solid substrates typically include materials applicable in medical, biochemical, or biological assays, such as substrates used in apheresis, column chromatography for the purification or separation of biological or organic molecules, and ELISA assays. Solid substrates may be porous or non-porous.

[0023]

[0024] Solid substrates for immobilizing affinity matrices that bind to the AAV antibody of the present invention are known in the art. Non-limiting examples of solid substrates include polymers such as polysaccharides. Non-limiting examples of polysaccharides include high molecular weight polysaccharides, in particular polysaccharides having a molecular weight of 100 kDa or higher, such as agarose. Agarose may be in particulate form, and the particulate form may optionally be crosslinked. A specific non-limiting example of agarose is Sepharose (trademark). Yet another non-limiting example of a polysaccharide is cellulose, which may optionally be crosslinked.

[0024]

[0025] Other suitable polymers as substrates include, for example, carboxylated polystyrene. The solid substrate may be provided in the form of magnetic beads. Glass is also a suitable substrate material.

[0025]

[0026] Any suitable blood or plasma filtration column or system can be configured for the apheresis column or apheresis method disclosed herein. Non-limiting examples include the column described in U.S. Patent No. 4,619,639, membrane filtration systems (e.g., MDF) used with suitable particles, surfaces, or substrates, and the PlasmaFlo® OP-05(W)L and RheoFilter® AR2000 blood filters manufactured by Asahi Medical Company, Ltd. in Japan.

[0026]

[0027] Apheresis columns or the methods of the present invention for the removal, depletion, capture, and / or inactivation of AAV-binding antibodies from the target blood may be performed once or repeatedly as necessary to achieve the desired results. In some embodiments, the apheresis method may be performed daily, every two days, every three days, every four days, once a week, every two weeks, twice a month, once a month, every two months, or a combination thereof, in an effort to obtain a beneficial therapeutic effect.

[0027]

[0028] In certain embodiments, the AAV gene therapy vector described herein is administered after the AAV-binding antibody has been removed, depleted, captured, and / or inactivated from the blood product of interest. The AAV gene therapy vector may be administered to the subject immediately after the apheresis method. In some embodiments, the AAV gene therapy vector is administered within at least 1 minute, at least 10 minutes, at least 20 minutes, at least 60 minutes, at least 1 hour, at least 4 hours, at least 8 minutes, at least 12 minutes, or at least 24 hours after the AAV-binding antibody has been removed, depleted, captured, and / or inactivated from the blood product of interest. In some embodiments, the AAV gene therapy vector is administered within 1 minute to 24 hours, 1 minute to 8 hours, or 1 minute to 4 hours after the AAV-binding antibody has been removed, depleted, captured, and / or inactivated from the blood product of interest.

[0028]

[0029] In certain embodiments, the AAV-conjugated antibody affinity matrix includes covalent bonds that link the AAV-conjugated antibody affinity matrix to the substrate. In some embodiments, preferred covalent bonds include peptide bonds.

[0029]

[0030] In certain embodiments, the AAV-conjugated antibody affinity matrix includes a linker for linking the AAV-conjugated antibody affinity matrix to a substrate. The linker can provide a mechanism for covalently attaching the AAV-conjugated antibody affinity matrix to the substrate. Any suitable linker can be used in the compositions or methods disclosed herein. Any suitable covalent bond or linker can be used to link the AAV-conjugated antibody affinity matrix to the substrate.

[0030]

[0031] In some embodiments, the linker comprises one or more amino acids, such as a peptide linker. The peptide linker may comprise any preferred number of amino acids. In some embodiments, the peptide linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acids. In certain embodiments, the peptide linker comprises 1 to 50, 1 to 20, 1 to 10, or 1 to 5 amino acids. In some embodiments, the peptide linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Non-limiting examples of amino acids and peptide linkers include one or more glycine residues, one or more serine residues, or combinations thereof. Additional preferred linkers include one or more carbons, silanes, thiols, phosphonic acids, and polyethylene glycol (PEG), or combinations thereof.

[0031]

[0032] A covalent bond can be attached to the N-terminus or C-terminus of the AAV-conjugated antibody affinity matrix. A linker can be attached to the N-terminus or C-terminus of the AAV-conjugated antibody affinity matrix.

[0032]

[0033] Methods of linking two or more molecules using covalent bonds or linkers are well known in the art and are sometimes referred to as "crosslinking." Non-limiting examples of crosslinking include amines reacting with N-hydroxysuccinimide (NHS) esters, imide esters, pentafluorophenyl (PFP) esters, hydroxymethylphosphine, oxiranes or any other carbonyl compounds; carboxyls reacting with carbodiimides; sulfhydryls reacting with maleimides, haloacetyls, pyridyl disulfide, and / or vinyl sulfones; aldehydes reacting with hydrazines; any non-selective groups reacting with diazirines and / or aryl azides; hydroxyls reacting with isocyanates; hydroxylamines reacting with carbonyl compounds; and combinations thereof.

[0033]

[0034] The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector), or other vehicle that can be manipulated by insertion or integration of nucleic acids. Vectors can be used for gene manipulation (i.e., "cloning vectors") to introduce / transfer polynucleotides into cells and cause the inserted polynucleotides to be transcribed or translated in the cells. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence along with the necessary regulatory regions required for expression in a host cell. The nucleic acid sequence of a vector generally contains at least an origin of replication for reproduction in cells and optionally contains additional elements, such as heterologous polynucleotide sequences, expression regulatory elements (e.g., promoters, enhancers), introns, reverse-terminal repeats (ITRs), selection markers (e.g., antibiotic resistance), and polyadenylation signals.

[0034]

[0035] Viral vectors are derived from or based on one or more nucleic acid elements that make up the viral genome. A specific type of viral vector is the adeno-associated virus (AAV) vector.

[0035]

[0036] Beyond the vector modifier, such as recombinant AAV vectors, the term “recombinant” as a sequence modifier, such as recombinant polynucleotides and polypeptides, generally means that the composition has been manipulated (i.e., engineered) in a manner not occurring naturally. A specific example of a recombinant AAV vector is when a nucleic acid sequence not normally present in the wild-type AAV genome is inserted into the AAV genome. The term “recombinant” is not necessarily used herein in relation to sequences such as polynucleotides other than AAV vectors, but recombinant forms involving heterologous polynucleotides are expressly included, in any such omission.

[0036]

[0037] A "recombinant AAV vector" or "rAAV" is derived from the wild-type AAV genome by using molecular methods to remove the wild-type genome and replacing it with a non-natural nucleic acid sequence, known as a heterologous nucleic acid. Typically, one or both of the reverse-terminal repeat (ITR) sequences of the AAV genome are retained for AAV. rAAV is distinguished from the AAV genome because all or part of the AAV genome is replaced with a non-natural sequence with respect to the AAV genome nucleic acid. The incorporation of a non-natural or heterologous sequence defines an AAV vector as a "recombinant" vector, which can therefore be called an "rAAV vector."

[0037]

[0038] rAAV sequences can be packaged (hereinafter referred to as “particles”) for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo. When recombinant AAV vector sequences are capsidized or packaged into AAV particles, the particles may also be referred to as “rAAV vectors” or “rAAV particles.” Such rAAV particles contain proteins that capsidize or package the vector genome. In the case of AAV, these are referred to as capsid proteins.

[0038]

[0039] The AAV vector “genome” refers to the portion of the recombinant plasmid sequence that is ultimately packaged or capsidized to form a viral (e.g., AAV) particle. When a recombinant plasmid is used to construct or manufacture a recombinant vector, the vector genome does not include the portion of the “plasmid” that does 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, which are necessary processes for reproduction and the manufacture of recombinant viruses, but is not itself packaged or capsidized into a viral (e.g., AAV) particle. Therefore, the vector “genome” refers to the nucleic acid that is packaged or capsidized by the virus (e.g., AAV).

[0039]

[0040] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to all forms of nucleic acids and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, as well as splicing or unsplicing mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acids).

[0040]

[0041] Polynucleotides may be single-stranded, double-stranded, or triplex, linear, or cyclic, and may be of any length. In discussions of polynucleotides, the sequence or structure of a particular polynucleotide may be described herein in accordance with the convention of describing the sequence from 5' to 3'.

[0041]

[0042] "Transgene" is used herein for convenience to refer to a heterogeneous nucleic acid that is intended or introduced into a cell or organism. Transgenes include any heterogeneous nucleic acid, such as a gene that codes for a polypeptide or protein, or a gene that codes for an inhibitory RNA.

[0042]

[0043] The terms "transduce" and their grammatical variations refer to the introduction of a molecule, such as an rAAV vector, into a cell or host organism. The heterologous nucleic acid / transgene may or may not be integrated into the genomic nucleic acid of the recipient cell. The introduced heterologous nucleic acid may also be extrachromosomal in the recipient cell or host organism, or may be transient only.

[0043]

[0044] A "transduced cell" is a cell into which a transgene has been introduced. Therefore, a "transduced" cell (e.g., in mammals, e.g., a cell, tissue, or organ cell) refers to a genetic change in a cell after the incorporation of an exogenous molecule, e.g., nucleic acid (e.g., a transgene), into the cell. Thus, a "transduced" cell is a cell into which an exogenous nucleic acid has been introduced, or its offspring. One or more cells can be propagated and the introduced protein can be expressed or the nucleic acid can be transcribed. For the use and methods of gene therapy, one or more transduced cells may be present in the subject.

[0044]

[0045] A “regulatory element” refers to one or more nucleic acid sequences that affect the expression of a operably linked nucleic acid. Regulatory elements include those described herein, such as promoters and enhancers. A vector sequence containing an AAV vector may contain one or more “regulatory elements.” Typically, such elements are included to facilitate the transcription and, where appropriate, translation of the appropriate heterologous polynucleotide (e.g., promoters, enhancers, splicing signals for introns, maintenance of the correct reading frame of a gene to enable in-frame translation of mRNA, and stop codons). Such elements typically act in the cis and are referred to as “cis-acting” elements, but they may also act in the trans.

[0045]

[0046] Expression regulation can occur at levels such as transcription, translation, splicing, and message stability. Typically, regulatory elements that modulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the nucleic acid being transcribed. Regulatory elements can also be located at the 3' end (i.e., "downstream") of the sequence being transcribed or within the transcript (e.g., in an intron). Regulatory elements can be located adjacent to or far from the sequence being transcribed (e.g., 1-10, 10-25, 25-50, 50-100, 100-500, or more nucleotides from the polynucleotide), even at considerable distances. Nevertheless, due to the length limitations of AAV vectors, regulatory elements are typically located within 1-1000 nucleotides from the nucleic acid being transcribed.

[0046]

[0047] Functionally, the expression of operably linked nucleic acids is at least partially regulated by elements (e.g., promoters) that modulate the transcription of the nucleic acid and, where appropriate, the translation of the transcript. A specific example of an expression regulatory element is a promoter, typically located at the 5' of the nucleic acid sequence being transcribed. Promoters increase the amount expressed from a typically operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0047]

[0048] As used herein, "enhancer" may refer to a sequence located adjacent to a heterologous nucleic acid. Enhancer elements typically function upstream of promoter elements, but can also be located downstream or within the sequence. Therefore, enhancer elements can be located 10–50 base pairs, 50–100 base pairs, 100–200 base pairs, 200–300 base pairs, or more than 10–50 base pairs upstream or downstream of the heterologous nucleic acid sequence. Enhancer elements typically increase the expression of the operably linked nucleic acid beyond the expression conferred by the promoter element.

[0048]

[0049] Expression constructs may include regulatory elements that act to promote expression in specific cell or tissue types. Examples of regulatory elements (e.g., promoters) that are active in specific tissues or cell types are referred to herein as “tissue-specific regulatory elements / promoters.” Tissue-specific regulatory elements are typically active in specific cells or tissues (e.g., the liver). Regulatory elements are typically active in specific cells, tissues, or organs because they are recognized by transcription-activating proteins or other transcriptional regulators specific to those cell, tissue, or organ types. Such regulatory elements are known to those skilled in the art (see, for example, Sambrook et al. (1989) and Ausubel et al. (1992)).

[0049]

[0050] The incorporation of tissue-specific regulatory elements in expression constructs provides at least partial tissue-specificity for the expression of heterologous nucleic acids encoding proteins or inhibitory RNAs. Examples of promoters active in the liver include, in particular, the TTR promoter, the human alpha-1 antitrypsin (hAAT) promoter; albumin, Miyatake, et al. J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig, et al., Gene Ther. 3:1002-9 (1996); and alpha-fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14 (1996). Examples of enhancers active in the liver include apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0050]

[0051] Expression regulatory elements include ubiquitous or promiscuous promoters / enhancers that can promote polynucleotide expression in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) initial promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence and other viral promoters / enhancers active in various mammalian cell types, or synthetic elements not found in nature (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerol kinase (PGK) promoter.

[0051]

[0052] Expression regulatory elements can also confuse expression in a modulated manner, i.e., a signal or stimulus increases or decreases the expression of an operably linked heterologous polynucleotide. A modulated element that increases the expression of an operably linked polynucleotide in response to a signal or stimulus is also referred to as an "inducible element" (i.e., signal-induced). Specific examples include, but are not limited to, hormone (e.g., steroid)-inducible promoters. Typically, the amount of increase or decrease confeded by such an element is proportional to the amount of signal or stimulus present; the greater the signal or stimulus, the greater the increase or decrease in expression. Specific non-limiting examples include the zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (International Publication No. 98 / 10088); the tetracycline-inhibiting system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen, et al., Science. 268:1766-1769 (1995); see also Harvey, et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); and the RU486-inducible system (Wang, et al., Nat. Biotech. 15:239-243 (1997) and Wang, et al., Gene Examples include Ther. 4:432-441 (1997); and the rapamycin-inducible system (Magari, et al., J. Clin. Invest. 100:2865-2872 (1997); Rivera, et al., Nat. Medicine. 2:1028-1032 (1996)). Other modulable regulatory elements that may be useful in this context are those regulated by specific physiological conditions, such as temperature, acute phase, and development.

[0052]

[0053] Expression regulatory elements also include (one or more) native elements for heterologous polynucleotides. Native regulatory elements (e.g., promoters) may be used when it is desired that the expression of heterologous polynucleotides mimic native expression. Native elements may be used when the expression of heterologous polynucleotides should be regulated transiently, developmentally, in a tissue-specific manner, or in response to a specific transcriptional stimulus. Other native expression regulatory elements, such as introns, polyadenylation sites, or Kozak consensus sequences, may also be used.

[0053]

[0054] The term "operably linked" means that regulatory sequences necessary for the expression of a nucleic acid sequence are positioned appropriately relative to the sequence to result in its expression. This same definition may apply to the configuration of a nucleic acid sequence and transcriptional regulatory elements (e.g., promoters, enhancers, and stop elements) in an expression vector, such as an rAAV vector.

[0054]

[0055] In the example of an expression regulatory element operably linked to a nucleic acid, the relationship is such that the regulatory element modulates the expression of the nucleic acid. More specifically, for example, two operably linked DNA sequences mean that the two DNA sequences are arranged (cis or trans) in a relationship such that at least one of the DNA sequences exerts a physiological effect on the other sequence.

[0055]

[0056] Therefore, additional elements for the vector include, but are not limited to, expression regulatory (e.g., promoter / enhancer) elements, transcription termination signals or stop codons, 5' or 3' untranslated regions adjacent to sequences such as one or more copies of an AAV ITR sequence (e.g., polyadenylated (polyA) sequences), or introns.

[0056]

[0057] Further elements include, for example, filler or stuffer polynucleotide sequences, e.g., filler or stuffer polynucleotide sequences to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically tolerate DNA inserts having a size range of approximately 4 kb to approximately 5.2 kb, or slightly larger. Therefore, for shorter sequences, stuffers or fillers are included to adjust the length to near or up to the normal size of the viral genome sequence that is acceptable for packaging the AAV vector into viral particles. In various embodiments, the filler / stuffer nucleic acid sequence is an untranslated (non-protein-coding) segment of the nucleic acid. For nucleic acid sequences less than 4.7 kb, the filler or stuffer polynucleotide sequence has a total length of approximately 3.0 to 5.5 kb, or approximately 4.0 to 5.0 kb, or approximately 4.3 to 4.8 kb when combined with the sequence (e.g., inserted into the vector).

[0057]

[0058] When used as a modifier for a composition, the term “isolated” means that the composition is either artificially created or completely or at least partially isolated from its naturally occurring in vivo environment. Generally, an isolated composition is substantially free from one or more materials that normally coexist naturally, such as one or more proteins, nucleic acids, lipids, carbohydrates, or cell membranes.

[0058]

[0059] The term “isolated” does not exclude artificially manufactured combinations, such as rAAV sequences or AAV vector genomes and rAAV particles that package or capsid pharmaceutical formulations. The term “isolated” also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, multimer / oligomer, modified (e.g., phosphorylated, glycosylated, lipidized) or derivatized forms, or forms expressed in artificially manufactured host cells.

[0059]

[0060] When referring to a specific nucleotide or amino acid sequence, the phrase "essentially derived from" means that the sequence possesses the characteristics of a given reference sequence. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself as well as molecular modifications that do not affect the sequence's fundamental and novel characteristics.

[0060]

[0061] The terms “identity,” “homology,” and their grammatical variations mean that two or more referenced entities are the same if they are “aligned” sequences. For example, if two protein sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. If two nucleic acid sequences are identical, they have the same nucleic acid sequence, at least within the referenced region or portion. Identity may extend across a defined section (region or domain) of a sequence.

[0061]

[0062] A “compartment” or “region” of identity refers to the same part of two or more referenced entities. Therefore, if two protein or nucleic acid sequences are identical across one or more sequence compartments or regions, they share identity within that region. An “aligned” sequence refers to multiple protein (amino acid) or nucleic acid sequences that often contain deletions or corrections of additional bases or amino acids (gaps) compared to a reference sequence.

[0062]

[0063] Identity may extend over the entire length or a portion of the sequence. In certain embodiments, the length of sequences sharing identity percentage is 2, 3, 4, 5 or more consecutive amino acids or nucleic acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive nucleic acids or amino acids. In additional embodiments, the length of sequences sharing identity is 21 or more consecutive amino acids or nucleic acids, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 consecutive amino acids or nucleic acids. In further embodiments, the length of sequences sharing identity is 41 or more consecutive amino acids or nucleic acids, e.g., 42, 43, 44, 45, 45, 47, 48, 49, 50 consecutive amino acids or nucleic acids. In a further embodiment, the length of the sequence sharing identity is 50 or more consecutive amino acids or nucleic acids, for example, consecutive amino acids or nucleic acids of 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-500, 500-1,000, etc.

[0063]

[0064] The degree of identity (homology) or "identity percentage" between two sequences can be verified using computer programs and / or mathematical algorithms. For the purposes of this invention, nucleic acid sequence comparisons are performed using the GCG Wisconsin Package version 9.1, available from the Genetic Computer Group in Madison, Wisconsin. For convenience, the default parameters specified by the program (gap generation penalty = 12, gap extension penalty = 4) are intended to be used in this invention to compare sequence identity. Alternatively, the Blastn 2.0 program provided by the National Center for Biotechnology Information (found on the worldwide web at ncbi.nlm.nih.gov / blast / ; Altschul et al., 1990, J Mol Biol 215:403-410), which uses gap alignment with default parameters, may be used to determine the levels of identity and similarity between nucleic acid sequences and amino acid sequences. For polypeptide sequence comparison, the BLASTP algorithm is typically used in combination with scoring matrices such as PAM100, PAM250, BLOSUM 62, or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantify the degree of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs for quantifying protein structure similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0064]

[0065] Nucleic acid molecules, expression vectors (e.g., AAV vector genomes), plasmids, and heterologous nucleic acids may be prepared using recombinant DNA techniques. The availability of nucleotide sequence information allows for the preparation of isolated nucleic acid molecules of the present invention by various means. For example, nucleic acid sequences encoding therapeutic proteins can be prepared using various standard cloning and recombinant DNA techniques via cell expression or in vitro translation and chemosynthesis techniques. The purity of polynucleotides can be determined through sequencing and gel electrophoresis, etc. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to, (1) hybridization of genomic DNA or cDNA libraries with probes for detecting homologous nucleotide sequences; (2) antibody screening for detecting polypeptides with shared structural features, for example, using expression libraries; (3) polymerase chain reaction (PCR) of genomic DNA or cDNA using primers that can anneal to the nucleic acid sequence of interest; (4) computer search of sequence databases for relevant sequences; and (5) differential screening of subset nucleic acid libraries.

[0065]

[0066] The nucleic acid may be maintained as DNA in any convenient cloning vector. For example, the clone can be maintained in a plasmid cloning / expression vector such as pBluescript (Stratagene, La Jolla, CA) which grows in suitable E. coli host cells. Alternatively, the nucleic acid may be maintained in a vector suitable for expression in mammalian cells, such as an AAV vector.

[0066]

[0067] As disclosed herein, an rAAV vector (actor) may optionally include regulatory elements necessary for the expression of a heterologous nucleic acid in a cell arranged in a manner that enables the expression of the encoded protein in the host cell. Such regulatory elements required for expression include, but are not limited to, promoter sequences, enhancer sequences, and transcription initiation sequences described herein and known to those skilled in the art.

[0067]

[0068] The methods and uses of the present invention involve delivering (introducing) nucleic acids (transgenes) to host cells, including dividing and / or non-dividing cells. The nucleic acids, rAAV vectors, methods, uses, and pharmaceutical formulations of the present invention are additionally useful in methods of delivering, administering, or providing a sequence encoded by heterologous nucleic acids to a subject in need as a therapeutic method. In this method, the nucleic acid is transcribed, and the protein or inhibitory nucleic acid may be produced in vivo in the subject. The subject may benefit from or need the protein or inhibitory nucleic acid for any reason, such as having a protein deficiency, or the production of the protein or inhibitory nucleic acid in the subject may confer some therapeutic effect as a therapeutic method. For example, inhibitory nucleic acids can reduce the expression or transcription of abnormal or harmful proteins expressed in a subject where obvious or harmful proteins are causing a disease or disorder such as a neurological disorder or impairment.

[0068]

[0069] rAAV vectors containing AAV genomes with heterologous nucleic acids enable the treatment of hereditary diseases. For deficiency diseases, gene transfer can be used to deliver normal genes to affected tissues for replacement therapy, and antisense mutations can be used to create animal models for the disease. For imbalanced conditions, gene transfer can be used to create the disease in a model system, which can then be used in efforts to combat the condition. Site-directed integration of nucleic acid sequences to correct defects is also possible.

[0069]

[0070] In various embodiments, rAAV vectors containing an AAV genome with heterologous nucleic acids can be used, for example, as therapeutic and / or prophylactic agents (proteins or nucleic acids). In certain embodiments, the heterologous nucleic acids encode proteins that can modulate the blood coagulation cascade.

[0070]

[0071] For example, encoded FVIII or FVIII-BDD may have coagulation activity similar to wild-type FVIII, or it may have altered coagulation activity compared to wild-type FVII. Administration of an FVIII or FVIII-BDD coding rAAV vector to a patient results in the expression of FVIII or FVIII-BDD proteins that work to normalize the coagulation cascade.

[0071]

[0072] In additional embodiments, the heterologous nucleic acid encodes a protein (enzyme) that can inhibit or reduce glycogen accumulation, prevent glycogen accumulation, or degrade glycogen. For example, the encoded GAA may have activity similar to wild-type GAA. Administration of a GAA-coding rAAV vector to a patient with Pompe disease works to inhibit or reduce glycogen accumulation, prevent glycogen accumulation, or degrade glycogen, which then results in the expression of a GAA protein that can reduce or lessen one or more adverse effects of Pompe disease.

[0072]

[0073] rAAV vectors can be administered alone or in combination with other molecules. According to the present invention, rAAV vectors or combinations of therapeutic agents can be administered to patients alone or in pharmaceutically acceptable or biocompatible compositions.

[0073]

[0074] Direct delivery of rAAV vectors or ex vivo transduction of human cells followed by injection into the body results in the expression of heterologous nucleic acids, thereby exerting beneficial therapeutic effects against congestion. In the context of blood coagulation factors such as factor VIII, administration enhances procoagulant activity. In the context of enzymes such as GAA, administration reduces the amount or accumulation of glycogen, prevents glycogen accumulation, or breaks down glycogen. This, in turn, can reduce or diminish one or more adverse effects of Pompe disease, such as promoting or improving muscle tone and / or muscle strength, and / or reducing or decreasing hepatomegaly.

[0074]

[0075] In addition to recombinant AAV vectors, the methods and their use include any viral strain or serotype. As a non-limiting example, recombinant AAV vectors may be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10, or AAV-2i8. Such vectors may be based on the same strain or serotype (or subgroup or variant), or they may be different from each other. As a non-limiting example, recombinant AAV vectors based on a particular serotype genome may be identical to the serotype of the capsid protein packaging the vector. Furthermore, recombinant AAV vector genomes may be based on an AAV serotype genome distinct from the serotype of the AAV capsid protein packaging the vector. For example, the AAV vector genome may be based on AAV2, while at least one of the three capsid proteins may be, for example, SEQ ID NO: 1, SEQ ID NO: 2, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 or a variant thereof.

[0075]

[0076] In certain embodiments, the adeno-associated virus (AAV) vectors include, for example, variants thereof (e.g., capsid variants, e.g., amino acid insertions, additions, substitutions, and deletions) as described in International Publication No. 2013 / 158879 (International Application PCT / US2013 / 037170), International Publication No. 2015 / 013313 (International Application PCT / US2014 / 047670), and U.S. Patent Application Publication No. 2013 / 0059732 (U.S. Patent No. 9,169,299; disclosing LK01, LK02, LK03, etc.).

[0076]

[0077] As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinction is determined on the basis that there is no cross-reactivity between antibodies against one AAV compared to another AAV. Such 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). Despite the possibility that an AAV variant may not be serologically distinct from a reference AAV or other AAV serotype, they differ from the reference or other AAV serotype in at least one nucleotide or amino acid residue.

[0077]

[0078] Under the traditional definition, a serotype means that the virus of interest has been tested for neutralizing activity against sera specific to all existing and characterized serotypes, and no antibodies neutralizing the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, they may or may not have serological differences from any existing serotype currently in use. Therefore, if a new virus (e.g., AAV) does not have serological differences, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. Often, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are a different serotype according to the traditional definition of serotype. Therefore, for simplicity and to avoid repetition, the term “serotype” broadly refers to both serologically distinct viruses (e.g., AAV) and viruses that are not serologically distinct (e.g., AAV) that may fall into a subgroup or variant of a given serotype.

[0078]

[0079] As described herein, AAV capsid proteins and the nucleic acids encoding capsid proteins exhibit less than 100% sequence identity to a reference or parental AAV serotype such as SEQ ID NO: 1, SEQ ID NO: 2, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, or AAV-2i8, but are distinct from and not identical to known AAV genes or proteins such as SEQ ID NO: 1, SEQ ID NO: 2, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8. In one embodiment, the AAV capsid protein contains or consists of a sequence that is identical to, or consists of, at least 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, and up to 99.9% of, a reference or parent AAV capsid protein such as SEQ ID NO: 1, SEQ ID NO: 2, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8.

[0079]

[0080] In certain embodiments, the modified AAV capsid protein has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions. In certain embodiments, the modified AAV capsid protein has a peptide insertion length of 2, 3, 4, 5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-50 or 50-60 amino acids.

[0080]

[0081] The rAAV vector may be administered to the patient by injection in a biocompatible carrier, for example, by intravenous injection. To increase molecular stability, the rAAV vector may optionally be encapsulated in liposomes or mixed with other phospholipids or micelles.

[0081]

[0082] rAAV vectors may be administered alone or in combination with other compositions, agents, drugs, or biological products. Therefore, rAAV vectors can be incorporated into pharmaceutical compositions, both alone and together with other compositions, agents, drugs, or biological products (proteins). Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo.

[0082]

[0083] In certain embodiments, the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical substance that does not induce an adverse immune response in the individual being given the composition and can be administered without excessive toxicity.

[0083]

[0084] As used herein, the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a bioacceptable formulation, gas, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, a material that can be administered to a subject without causing a major undesirable biological effect. Such a pharmaceutical composition can therefore be used, for example, in the administration of nucleic acids, vectors, viral particles or proteins to a subject.

[0084]

[0085] Examples of pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, physiological saline, glycerol, sugar, and ethanol. Examples of pharmaceutically acceptable salts include mineral salts such as hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as acetate, propionate, malonate, and benzoate. Furthermore, auxiliary substances, such as wetting agents or emulsifiers, and pH buffering agents, may be present in such a vehicle.

[0085]

[0086] The pharmaceutical composition may also be provided as a salt, which can be formed with many acids, including, but not limited to, salts of hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, and succinic acid. The salts tend to be more soluble in aqueous or other protonating solvents than in their corresponding free base forms. In other cases, the preparation may be a lyophilized powder in the pH range of 4.5 to 5.5, containing any or all of the following: 1 to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, which is combined with a buffer before use.

[0086]

[0087] Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil in water or water in oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonic agents and absorption enhancers or retarders, which are suitable for pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may also contain suspending agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Auxiliary active compounds (e.g., preservatives, antimicrobial agents, antiviral agents and antifungal agents) may also be incorporated into the composition.

[0087]

[0088] Pharmaceutical compositions may be formulated to be compatible with specific routes of administration or delivery, as described herein or known to those skilled in the art. Accordingly, pharmaceutical compositions may include carriers, diluents, or excipients suitable for administration by various routes.

[0088]

[0089] Suitable compositions for parenteral administration include aqueous and non-aqueous solutions, suspensions, or emulsions of the active compound, which are typically sterile and may be isotonic with the blood of the intended recipient. Non-limiting examples include water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal oil, vegetable oil, or synthetic oil. Aqueous suspensions for injection may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0089]

[0090] Furthermore, the suspension of the active compound may be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound, enabling the preparation of a highly concentrated solution.

[0090]

[0091] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents include hydroxyl groups or other polar groups, such as alcohols like isopropyl alcohol; glycols like propylene glycol, polyethylene glycol, polypropylene glycol, and glycol ethers; glycerol; polyoxyethylene alcohol and polyoxyethylene fatty acid esters. Examples of adjuvants include surfactants such as soy lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0091]

[0092] After the pharmaceutical compositions are prepared, they can be placed in appropriate containers and labeled for processing. Such labeling can include indicating the dosage, frequency, and method of administration.

[0092]

[0093] The compositions, methods, and suitable pharmaceutical compositions and delivery systems for use of the present invention are known in the art (for example,Remington: The Science and Practice of Pharmacy (2003)20 th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA; Merck Index (1996)12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001)11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (See 1980, RL Juliano, ed., Oxford, NY, pp. 253–315).

[0093]

[0094] "Effective dose" or "sufficient dose" means an amount, either alone or in combination with one or more other compositions (such as therapeutic agents or immunosuppressants), treatments, protocols, or therapeutic regimens, that provides, in a single or multiple dose, a detectable response for any duration (long-term or short-term), any measurable or detectable degree of expected or desired outcome or benefit to a subject for any duration (e.g., minutes, hours, days, months, years, or during a cure).

[0094]

[0095] The dosage may vary and depends on the type of disease being treated, its onset, progression, severity, frequency, duration, or probability, the desired clinical endpoint of the subject, prior or concurrent treatments, general health status, age, gender, race or immunological eligibility, and other factors understood by those skilled in the art. The dosage, number of doses, frequency, or duration may be increased or decreased proportionally as indicated by any adverse side effects, complications, or other risk factors of the treatment or therapy and the condition of the subject. Those skilled in the art understand the factors that may affect the dosage and timing required to provide an amount sufficient to achieve a therapeutic or prophylactic benefit.

[0095]

[0096] The dosage to achieve a therapeutic effect, e.g., the dosage of vector genome per kilogram of body weight (vg / kg), varies based on several factors, including the route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease being treated, any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein, but is not limited thereto. Those skilled in the art can determine the dosage range of rAAV / vector genome for treating patients with a specific disease or disorder based on the above factors and other factors.

[0096]

[0097] Generally, the dosage is at least 1×10 8 vector genomes (vg / kg) per kilogram of the subject's body weight or more to achieve a therapeutic effect, e.g., 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or 1×10 14 , or more vector genomes (vg / kg), and is within the range. In mice, it is 1×10 10 ~1×10 11 vg / kg, and in dogs, it is 1×10 12 ~1×10 13rAAV doses within the range of vg / kg were effective. For example, a dose of 6 × 10⁻⁶ 12 It may also be lower, such as a dose less than vg / kg. More specifically, 5 × 10 11 vg / kg or 1 × 10⁻⁶ 12 The dosage is in 1g / kg.

[0097]

[0098] The rAAV vector dose may typically be at the lower end of the dose spectrum, such that there is no significant immune response to the heterologous nucleic acid sequence, encoded protein or inhibitory nucleic acid, or the rAAV vector. More specifically, up to 6 × 10⁻⁶ 12 The value is vg / kg, but less than that, for example, approximately 5 × 10 11 ~Approx. 5×10 12 vg / kg, or more specifically, about 5 × 10 11 vg / kg or approximately 1 × 10⁻⁶ 12 The dosage is in 1g / kg.

[0098]

[0099] An effective or sufficient dose for treatment (e.g., for remission or to produce a therapeutic benefit or improvement) is typically effective to produce a response to one, more or all adverse symptoms, consequences, or complications of the disease, e.g., one or more adverse symptoms, disorders, illnesses, conditions, or complications caused by or related to the disease, to a measurable extent, but a satisfactory outcome is to reduce, mitigate, inhibit, suppress, limit, or control the progression or worsening of the disease.

[0099]

[0100] An effective or sufficient dose may or may not be provided as a single dose, but multiple doses may be required, and may or may not be provided alone or in combination with another composition (e.g., a drug), treatment, protocol or treatment regimen. For example, the dose may be increased proportionally as indicated by the needs of the subject, the type, condition and severity of the disease being treated, or any side effects of treatment (if any).

[0100]

[0101] In addition, an effective or sufficient dose does not need to be effective or sufficient when administered in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol or treatment regimen, for this reason, additional doses, amounts or durations, or additional compositions (e.g., drugs or agents), treatments, protocols or treatment regimens may be included to exceed such a dose in order to be considered effective or sufficient for a given subject. An effective dose may also be one that results in a reduction of the use of another treatment, treatment regimen or protocol, such as the administration of recombinant enzymes (e.g., GAA) for the treatment of enzyme deficiency (e.g., Pompe disease) or recombinant coagulation factor proteins (e.g., FVIII) for the treatment of coagulation disorders (e.g., hemophilia A).

[0101]

[0102] Accordingly, the methods and uses of the present invention also include, in particular, methods and uses that result in a reduction of the need for or use of another compound, agent, drug, treatment regimen, treatment protocol, method, or therapeutic agent. Accordingly, the present invention provides methods and uses that reduce the need for or use of another treatment or therapy.

[0102]

[0103] An effective or sufficient dose does not need to be effective in each and every subject being treated, nor does it need to be effective in the majority of subjects being treated within a given group or population. An effective or sufficient dose refers to efficacy or sufficiency in a specific subject, not in a group or general population. As is typical with such methods, some subjects may exhibit a greater or lesser response, or no response at all, to a given treatment method or use.

[0103]

[0104] The term "remission" means a detectable or measurable improvement in the disease or its symptoms, or in the underlying cellular response. Detectable or measurable improvements include subjective or objective reduction, decrease, inhibition, suppression, limitation, or control of the presence, frequency, severity, progression, or duration of the disease, or complications caused by or associated with the disease; or improvement in the symptoms or underlying causes or consequences of the disease; or regression of the disease.

[0104]

[0105] For Pompe disease, the effective dose is, for example, the amount of GAA that inhibits or reduces glycogen production or accumulation, promotes or increases glycogen breakdown or removal, or improves muscle tone and / or muscle strength in the subject. For hemophilia A, the effective dose is, for example, the amount that reduces the frequency or severity of acute bleeding in the subject, or, for example, the amount that reduces clotting time as measured by a coagulation assay.

[0105]

[0106] The therapeutic dose depends, among other factors, particularly the patient's age and overall condition, and the severity of the disease or disability. The effective therapeutic dose in humans falls within a relatively wide range, which may be determined by the physician based on the individual patient's response.

[0106]

[0107] A composition such as a pharmaceutical composition may be delivered to a subject to enable the production of an encoded protein or inhibitory nucleic acid. In certain embodiments, the pharmaceutical composition contains sufficient genetic material to enable the recipient to produce a therapeutically effective amount of the protein or inhibitory nucleic acid in the subject.

[0107]

[0108] The composition may be administered alone. In certain embodiments, recombinant AAV particles produce a therapeutic effect without immunosuppressants. The therapeutic effect is optionally sustained without the administration of immunosuppressants for a period of time of, for example, 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, or 30-50 days or longer, for example, 50-75, 75-100, 100-150, 150-200 days or longer. Thus, the therapeutic effect is produced over a period of time.

[0108]

[0109] The composition may be administered in combination with at least one other agent. In certain embodiments, the rAAV vector is administered substantially simultaneously with or after the administration of the rAAV vector in combination with one or more immunosuppressants. In certain embodiments, the administration of an immunosuppressant after the administration of the rAAV vector is, for example, 1-12, 12-24, or 24-48 hours, or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50 days, or more than 50 days. Such administration of an immunosuppressant after a certain period after the administration of the rAAV vector occurs when there is a decrease in the encoded protein or inhibitory nucleic acid after the initial expression level over a certain period after the rAAV vector, for example, 20-25, 25-30, 30-50, 50-75, 75-100, 100-150, 150-200 days, or more than 200 days.

[0109]

[0110] In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid. In certain embodiments, the immunosuppressant is cyclosporine (e.g., cyclosporine A), mycophenolate, rituximab, or derivatives thereof. Additional specific agents include stabilizing compounds.

[0110]

[0111] The composition may be formulated and / or administered in any sterile, biocompatible pharmaceutical carrier, including but not limited to physiological saline, buffered physiological saline, dextrose, and water. The composition may be formulated and / or administered to the patient alone or in combination with other agents that affect congestion (e.g., cofactors).

[0111]

[0112] The methods and uses of the present invention include delivery and administration systemically, locally, or topically, or by any route, such as injection or infusion. In vivo delivery of pharmaceutical compositions can generally be achieved via conventional syringe injection, but other delivery methods such as convection-enhanced delivery methods are also envisioned (see, for example, U.S. Patent No. 5,720,720). For example, the composition may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intrapleurally, intraarterially, orally, intrahepatically, via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application. Clinicians specializing in the treatment of patients with blood coagulation disorders can determine the optimal route for administration of AAV vectors based on a number of criteria, including, but not limited to, the patient's condition and the objective of treatment (e.g., enhancement or reduction of blood coagulation).

[0112]

[0113] The rAAV vectors, methods, and uses of the present invention can be combined with any compound, agent, drug, treatment, or other therapeutic regimen or protocol having the desired therapeutic, beneficial, additional, synergistic, or complementary activity or effect. Exemplary combination compositions and treatments include a second activator such as a biological agent (protein), agent (e.g., immunosuppressant), and drug. Such biological agents (proteins), agents, drugs, treatments, and therapies can be administered or carried out substantially concurrently with, or before, any other method or use of the present invention.

[0113]

[0114] Compounds, agents, drugs, treatments, or other therapeutic regimens or protocols may be administered as a combination composition or separately, for example, in parallel with, sequentially with, or after (before or after) the delivery or administration of nucleic acids, vectors, or rAAV particles. The present invention therefore provides combinations in which the methods or uses of the present invention are combined with any compound, agent, drug, therapeutic regimen, therapeutic protocol, method, therapeutic agent, or composition described herein or known to those skilled in the art. Compounds, agents, drugs, therapeutic regimens, therapeutic protocols, methods, therapeutic agents, or compositions may be administered to a subject before, substantially concurrently with, or after the administration of nucleic acids, vectors, or rAAV particles of the present invention.

[0114]

[0115] The present invention is useful in animals, including humans, and in veterinary applications. Preferred subjects, therefore, include mammals such as non-human mammals, in addition to humans. The term "subject" refers to animals, typically mammals, e.g., humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry, e.g., chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, neonates, infants, young children, and adult subjects. Subjects also include animal disease models of protein / enzyme deficiencies such as Pompe disease, blood coagulation disorders such as HemA, and others known to those skilled in the art, e.g., mice and other animal models.

[0115]

[0116] Appropriate subjects for treatment according to the present invention include subjects who produce insufficient amounts of functional gene products, have a deficiency in such gene products, are at risk of doing so, or produce abnormal, partially functional or nonfunctional gene products that may lead to disease. Appropriate subjects for treatment according to the present invention also include subjects who have or are at risk of producing abnormal or defective (mutant) gene products (proteins) that lead to disease, such that a reduction in the amount, expression, or function of the abnormal or defective (mutant) gene product (protein) leads to treatment of the disease, reduction of one or more symptoms, or remission of the disease.

[0116]

[0117] Subjects can be tested for immune responses, such as antibodies against AAV. Therefore, candidate subjects can be screened before treatment according to the method of the present invention. Subjects can also be tested for antibodies against AAV after treatment, and optionally, subjects can be monitored over a period after treatment. Subjects that produce AAV antibodies can be treated with immunosuppressants, or they can be administered one or more additional amounts of AAV vector.

[0117]

[0118] Suitable subjects for the treatment according to the present invention also include subjects who have antibodies against AAV or are at risk of producing such antibodies. Several techniques can be used to administer or deliver rAAV vectors to such subjects. For example, an empty AAV capsid (i.e., AAV lacking heterologous nucleic acid) can be delivered to bind to AAV antibodies in the subject, thereby enabling the introduction of an rAAV vector containing heterologous nucleic acid into the subject's cells.

[0118]

[0119] The ratio of AAV empty capsid to rAAV vector may be approximately 2:1 to 50:1, or approximately 2:1 to 25:1, or approximately 2:1 to 20:1, or approximately 2:1 to 15:1, or approximately 2:1 to 10:1. The ratio may also be approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0119]

[0120] The amount of AAV empty capsid to be administered may be adjusted based on the amount (titer) of AAV antibodies produced in a particular subject. The AAV empty capsid may be any serotype, for example, SEQ ID NO: 1, SEQ ID NO: 2, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8.

[0120]

[0121] Alternatively or additionally, the rAAV vector may be delivered by direct intramuscular injection (e.g., into one or more slow-twitch muscle fibers). Another alternative is to deliver the rAAV vector to the liver via the hepatic artery using a catheter introduced into the femoral artery. Non-surgical means such as endoscopic retrograde cholangiopancreatography (ERCP) can also be used to deliver the rAAV vector directly to the liver, thereby bypassing the bloodstream and AAV antibodies. Other duct systems, such as the submandibular duct, can also be used as portals for delivering the rAAV vector to subjects that generate or already have anti-AAV antibodies.

[0121]

[0122] The substance can be administered to or delivered in vivo to a subject before the onset of adverse symptoms, conditions, or complications caused by or associated with the disease. For example, screening (e.g., gene screening) can be used to identify such subjects as candidates for the composition, method, and use of the present invention. Such subjects would therefore include those that have been screened positive for insufficient amounts or deficiencies in functional gene products, or those that produce abnormal, partially functional, or nonfunctional gene products.

[0122]

[0123] Administration or in vivo delivery to a subject by the methods and uses of the present invention as disclosed herein can be carried out within 1-2, 2-4, 4-12, 12-24, or 24-72 hours after the subject has been identified as having a disease targeted for therapeutic purposes, having one or more symptoms of the disease, or has been screened and identified as positive as described herein, even if the subject does not have one or more symptoms of the disease. Naturally, the methods and uses of the present invention can be carried out within 1-7, 7-14, 14-24, 24-48, 48-64, or later days, months, or years after the subject has been identified as having a disease targeted for therapeutic purposes, having one or more symptoms of the disease, or has been screened and identified as positive as described herein, even if the subject does not have one or more symptoms of the disease.

[0123]

[0124] As used herein, “unit dosage form” refers to a physically separate unit suitable as a unit dose for a target to be treated, each unit containing a predetermined amount of a pharmaceutical carrier (excipient, diluent, vehicle, or filler) in an optional combination calculated to produce a desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms may be, for example, in ampoules and vials, which may contain a liquid composition or a composition in a freeze-dried or lyophilized state, and a sterile liquid carrier may be added, for example, before in vivo administration or delivery. Individual unit dosage forms may be contained in multi-dose kits or containers. rAAV particles and their pharmaceutical compositions may be packaged in one or more unit dosage forms to facilitate administration and ensure uniformity of the dose.

[0124]

[0125] A subject can be tested for protein activity to determine whether such a subject is suitable for treatment by the method of the present invention. A subject can also be tested for protein content according to the method of the present invention. Such a treated subject can be monitored regularly after treatment, for example, every 1-4 weeks, every 1-6 months, every 6-12 months, or every 1, 2, 3, 4, 5 or longer years.

[0125]

[0126] A subject can be tested for adverse responses to one or more liver enzymes to determine whether such a subject is suitable for treatment by the method of the present invention. Therefore, a candidate subject can be screened for the amount of one or more liver enzymes before treatment by the method of the present invention. A subject can also be tested for the amount of one or more liver enzymes after treatment by the method of the present invention. Such a treated subject can be monitored periodically for elevations of liver enzymes after treatment, for example, every 1-4 weeks, every 1-6 months, every 6-12 months, or every 1, 2, 3, 4, 5 or longer years.

[0126]

[0127] Examples of liver enzymes include alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), but other enzymes that indicate liver damage can also be monitored. Normal levels of these enzymes in circulation are typically defined as a range with an upper limit; enzyme levels above this upper limit are elevated and therefore considered indicators of liver damage. The normal range is based in part on the criteria used by the clinical trial laboratory performing the assay.

[0127]

[0128] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and materials may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0128]

[0129] All patents, patent applications, publications, and other references cited herein, as well as citations from GenBank and ATCC, are incorporated in their entirety by reference. In case of any conflict, the provisions of this specification, including definitions, shall prevail.

[0129]

[0130] Various terms relating to the biological molecules of the present invention are used above, as well as in this specification and in the claims.

[0130]

[0131] All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, the disclosed features are examples of a group of equivalent or similar features.

[0131]

[0132] As used herein, the singular forms "a," "and," and "the" refer to multiple objects unless the context explicitly indicates otherwise. For example, a reference to "a nucleic acid" includes multiple such nucleic acids, a reference to "a vector" includes multiple such vectors, and a reference to "a virus" or "a particle" includes multiple such viruses / particles.

[0132]

[0133] As used herein, all numbers or numerical ranges include integers and values ​​or decimals of integers within such ranges unless the context explicitly indicates otherwise. Therefore, to give an example, references to identity of 80% or higher include not only 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, but also 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, and so on.

[0133]

[0134] References to integers with "greater than" or "less than" include any number greater than or less than the reference number, respectively. For example, a reference to less than 100 includes all numbers decreasing down to 1, such as 99, 98, 97, etc., and less than 10 includes all numbers decreasing down to 1, such as 9, 8, 7, etc.

[0134]

[0135] Where used herein, all numbers or ranges include decimal values ​​and integers within such ranges and decimal integers within such ranges, unless the context explicitly indicates otherwise. For example, a reference to a numerical range such as 1–10 includes not only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, but also 1.1, 1.2, 1.3, 1.4, 1.5, and so on. A reference to a range of 1–50 therefore includes not only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and up to 50, but also 1.1, 1.2, 1.3, 1.4, 1.5, 2.1, 2.2, 2.3, 2.4, 2.5, and so on.

[0135]

[0136] A reference to a series of ranges includes the ranges that combine the values ​​of the boundaries of different ranges within the series. Therefore, to give an example, a reference to a series of ranges such as 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, and 750-850 would be 1-20, 1-30, 1-40, 1-50, 1-60, 10-30, 10-40, 10-50, and 10- This includes ranges such as 60, 10-70, 10-80, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 50-75, 50-100, 50-150, 50-200, 50-250, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 150-250, 150-300, 150-350, 150-400, 150-450, and 150-500.

[0136]

[0137] The present invention is generally disclosed herein using affirmative language to describe a number of embodiments and aspects. The present invention also particularly includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is excluded in whole or in part. For example, in certain embodiments or aspects of the present invention, materials and / or method steps are excluded. Thus, the present invention is generally not expressed herein in terms of what the present invention does not include, but nevertheless, aspects that are not expressly excluded in the present invention are disclosed herein.

[0137]

[0138] Several embodiments of the present invention have been described. Nevertheless, those skilled in the art can make various modifications and improvements to the invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate the concepts and are not intended to limit the scope of the claimed invention in any sense. [Examples]

[0138]

[0001] The following are representative non-limiting examples of affinity resins (GE Healthcare) that could be used to generate AAV capsid-specific antibody affinity matrices. The AAV capsid protein contains amino acids that can be crosslinked to the various chromatographic media described below. Equivalent and / or suitable materials from other manufacturers of affinity resins to which AAV capsids can be attached may also be used or manufactured as appropriate.

[0139] Example 1 CNBr-activated Sepharose 4 Fast Flow

[0002] CNBr-activated Sepharose 4 Fast Flow is a pre-activated chromatography medium for the linkage of large amino acid-containing ligands. A CNBr-activated BioProcess medium designed for the linkage of large amino acid-containing ligands. Rapid and efficient connection. This resin allows for the attachment of multiple protein ligands while minimizing ligand leakage. BioProcess media that are well-established in supported and certified methods for industrial applications.

[0140]

[0003] CNBr-activated Sepharose 4 Fast Flow is based on the established Sepharose Fast Flow platform. The resin consists of crosslinked 4% agarose beads pre-activated with cyanide bromide. CNBr-activated Sepharose 4 Fast Flow is designed for multi-point attachment of amino group-containing protein ligands.

[0141]

[0004] The preparation and use of affinity chromatography media by linking biomolecule-specific ligands to CNBr-activated Sepharose 4 Fast Flow is a widely used, well-documented approach involving a simple, rapid, and efficient linking procedure.

[0142]

[0005] CNBr-activated Sepharose 4 Fast Flow is available in a range of different bulk pack sizes and convenient pre-packed formats for easy scale-up and process development.

[0143]

[0006] As a member of the BioProcess media range, CNBr-activated Sepharose 4 Fast Flow meets industrial demand, with supply certainty and comprehensive technical and regulatory support.

[0144] Example 2 Activated Thiol Sepharose 4B

[0007] Activated thiol Sepharose 4B medium is a medium used for the reversible immobilization of thiol group-containing molecules under mild conditions. It is optimized for the immobilization of large molecules. Reversible linking of proteins and large biomolecules to Sepharose 4B using thiol groups via a glutathione spacer arm. The ligand is a mixed disulfide formed between 2,2'-dipyridyl disulfide and glutathione linked to CNBr-activated Sepharose 4B. It is well-suited for covalent chromatography of large molecules such as enzymes and nucleic acids. The gel also reacts with heavy metal ions, as well as alkyl and aryl halides. It undergoes addition reactions with compounds containing C=O, C=C, and N=N bonds. Thiol-containing proteins are separated from non-thiol-containing proteins.

[0145]

[0008] Activated thiol Sepharose 4B is a mixed disulfide formed between 2,2'-dipyridyl disulfide and glutathione linked to CNBr-activated Sepharose 4B. Activated thiol Sepharose 4B reacts with a thiol-containing solute under mild conditions to form a mixed disulfide. This reaction forms the basis for covalent chromatography and procedures for the immobilization of thiol-containing biomolecules.

[0146] Example 3 EAH Sepharose 4B

[0009] The EAH Sepharose pre-activation medium is used for linking compounds containing carboxyl groups to Sepharose 4B through linkage of a carbodiimide base via an 11-atom spacer arm. The stable linking of carboxyl groups to Sepharose 4B via an 11-atom hydrophilic spacer arm enables highly stable linkage of the carbodiimide base.

[0147] Example 4 Epoxy-activated Sepharose 6B

[0010] Epoxy-activated Sepharose 6B is a pre-activated medium for the immobilization of various ligands, including sugars, through the linkage of hydroxy, amino, or thiol groups of ligands to Sepharose 6B via a 12-atom hydrophilic spacer arm. Using epoxy-activated Sepharose 6B, sugars and other carbohydrates can be linked to hydroxyl groups via stable ether bonds.

[0148]

[0011] Epoxy-activated Sepharose 6B is a pre-activated medium for the immobilization of various ligands. Using epoxy-activated Sepharose 6B, sugars and other carbohydrates can be linked to hydroxyl groups via stable ether bonds. Other ligands can be linked via hydroxyl, amino, or thiol groups. The medium has long hydrophilic spacer arms, which makes it particularly suitable for the immobilization of small molecules. Epoxy-activated Sepharose 6B is formed by reacting Sepharose 6B with 1,4-bis(2,3-epoxy-propoxy-)butane.

[0149] Example 5 Purified AAV capsids can be GMP grade in essence.

[0012] A typical characteristic of AAV capsid proteins used in combination with a suitable resin or matrix to generate an AAV capsid affinity matrix is ​​purity. The generation of AAV in cell cultures is complex, and its separation from the abundant non-AAV components (impurities and contaminants) that are generated together is important. In particular, AAV capsid particles are purified from impurities derived from the manufacturing cells and cell culture medium. The presence of high levels of impurities in AAV capsid preparations used to generate affinity matrices results in lower efficiency and lower binding specificity when used in apheresis situations. One exemplary purification of AAV particles is under current Good Manufacturing Practices (GMP) for human parenteral products, because the assumed affinity matrix comes into contact with blood products of human interest during the plasmapheresis process.

[0150] Example 6 AAV capsid materials for affinity matrix fabrication

[0013] One typical form of capsid material for use in the generation of AAV capsid affinity matrices for plasmaferesis is the AAV "empty" capsid, which is an AAV particle lacking an transgene. Affinity matrix-bound AAV empty capsids are expected to present the same surface epitopes as the corresponding AAV vector.

[0151]

[0014] For example, highly purified empty capsids derived from AAV capsid variant SEQ ID NO: 1 or 2 can be linked to CNBr-activated Sepharose resin. In a human subject with severe hemophilia A and an antibody titer of 1:100 for AAV-SEQ ID NO: 1 or 2, sufficient plasmapheresis as described herein, using an AAV-binding antibody affinity matrix prepared with highly purified empty capsids for AAV-SEQ ID NO: 1 or 2, is expected to reduce the subject's antibody titer for AAV-SEQ ID NO: 1 or 2 to, for example, 1:1. Efficient gene transfer is achieved by subsequently treating the subject with AAV-SEQ ID NO: 1 or 2 expressing human coagulation factor VIII less than approximately 12 hours, or typically less than approximately 6 hours, after the completion of the plasmapheresis protocol, followed by the expression of therapeutically circulating FVIII. At higher initial AAV titers (higher than 1:100), post-plasmapheresis protocol treatment is performed earlier.

[0152]

[0015] Alternatively, empty capsids prepared for any other known AAV capsid serotype or capsid variant can be similarly used to reduce the antibody titer against that particular AAV capsid serotype or capsid variant, thereby enabling efficient gene transfer in the corresponding AAV vector expressing any therapeutic transgene.

[0153]

[0016] Furthermore, by alternative means, any AAV VP1, VP2, and VP3 capsid proteins from any naturally occurring AAV capsid serotype or synthetic AAV capsid variant can be used alone or in any combination of stoichiometric values ​​to reduce AAV antibody titers and enable efficient gene transfer in the corresponding viral vector expressing any therapeutic gene.

[0154] Example 7 Other viral vector materials for affinity matrix production

[0017] Alternatively, an antibody affinity matrix may be developed using other viral vectors or viral vector proteins from viruses that can be used to achieve gene transfer, and the method described herein may be carried out. Exemplaryly, viruses are from the following viral families: Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Papoviridae, Adenoviridae, Parvoviridae, Herpesviridae, Poxviridae, and Hepadnaviridae, which can be used to reduce the antibody titer of each virus to enable efficient gene transfer in the corresponding vector expressing any therapeutic gene.

[0155] Example 8 Re-administration and / or continuous administration of vectors for therapeutic benefit

[0018] In addition to the inhibition of therapeutic gene transfer by AAV vectors by naturally occurring antibodies in the human population, capsid-specific antibodies may increase after administration of the AAV vector (and other vectors as described herein). The use of AAV capsid affinity plasmapheresis can similarly reduce AAV capsid antibodies induced by prior administration of gene therapy vectors, enabling efficient gene transfer upon re-administration. This process can be carried out continuously over a long period to gradually increase the level of therapeutic gene expression in human subjects. For example, the blood level of coagulation factor FVIII after AAV-based gene transfer in infants with severe hemophilia A is thought to gradually decrease as the child grows. Regular re-administration during childhood and adolescence using the AAV capsid affinity plasmapheresis protocol disclosed herein can enable the maintenance of therapeutic levels of FVIII as the child grows and throughout adulthood.

[0156] Example 9 Calculation of antibody rebound rate IgG half-life (human) = 20 days The IgG concentration in human serum is in the range of 8-18 mg / mL, and 12 mg / mL is used. The exponential decay formula HID = No(1 / 2)t / t1 / 2 (where t is in days) Proof of the formula: H(0) = 12(1 / 2)0 / 20 = 12(1) = 12 mg / mL H(20)=12(1 / 2)20 / 20=12(1 / 2)=6mg / mL N= IgG loss 1 day = 24 hours N(t=1) = 12(1 / 2) 1 / 20 =12(0.5) 0.05 = 11.591 mg / mL 0.5 days = 12 hours N(0.5) = 12(1 / 2) 0.5 / 20 =12(0.5) 0.025 = 11.794 mg / mL 0.25 days = 6 hours N(0.25) = 12(1 / 2) 0.25 / 20 =12(0.5)0.0125 = 11.896 mg / mL =3 hours H(0.125)=12(1 / 2) 0.125 / 20 =12(0.5) 0.00625 = 11.948 mg / mL =1 hour N(0.042)=12(1 / 2)^0.042 / 20=12(0.5)^0.00208=11.983mg / mL A steady state of 12 mg / mL signifies an equal synthesis rate. IgG synthesis 24 hours 12 - 11.591 = 0.409 mg / m² 2 ÷12 = 3.41% 12 hours 12-11.794=0.206mg / m 2 ÷12 = 1.72% 6 hours 12-11.896=0.104mg / m 2 ÷12 = 0.87% 3 hours 12 - 11.948 = 0.052 mg / m² 2 ÷12 = 0.43% 1 hour 12-11.983=0.017mg / mL / 12=0.15% We assume that the synthesis rate is equally distributed across all IgG molecules. AAV capsid IgG, which starts at 1:100, is reduced to 1:1 by capsid plasmapheresis. 1:4.4 in 24 hours. 1:07 in 12 hours. 1:1.9 in 6 hours. 1:1.43 in 3 hours. 1 hour: 1.15 It rebounded.

[0157]

[0019] Table 1 shows a wider range of titer rebound rates as a function of initial AAV capsid IgG titers (i.e., 1:10, 1:230, 1:100, 1:300, 1:1000, 1:3000, 1:10000 as described above). In particular, Table 1 shows that AAV vectors for gene therapy can be administered to subjects with an AAV antibody titer of up to 1:1000 within approximately 1 hour after plasmaferesis, to subjects with an AAV antibody titer of up to 1:300 within approximately 3 hours after plasmaferesis, to subjects with an AAV antibody titer of up to 1:100 within approximately 6 hours after plasmaferesis, to subjects with an AAV antibody titer of up to 1:100 within approximately 12 hours after plasmaferesis, and to subjects with an AAV antibody titer of up to 1:30 within approximately 24 hours after plasmaferesis.

[0158] [Table 1]

[0159] Example 9 Representative AAV capsid (VP1) protein AAV-SPK VP1 capsid (SEQ ID NO: 1) 1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLD 61 KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ 121AKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDS 181ESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV 241 ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ 301RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSA 361HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFED 421VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNW 481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSS 541GVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNS 601QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADP 661PTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTE 721 GTYSEPRPIGTRYLTRNL AAV-LK03 VP1 Capsid (SEQ ID NO: 2) MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPD PQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPAD VFMVPN AELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

[0160]

[0020] The present invention may also be described as follows: 1. A method for treating a subject in need of treatment for a disease caused by loss of function or activity of a protein, comprising: (a) removing, reducing, depleting, inhibiting, inactivating or capturing AAV-binding antibodies from a blood product obtained from the subject by a process comprising apheresis; and (b) administering a certain amount of a recombinant adeno-associated virus (rAAV) vector comprising a heterologous polynucleotide encoding a protein or peptide that provides or assists the function or activity of the protein. 2. A method for treating a subject in need of treatment for a disease caused by the acquisition of function, activity or expression of a protein, comprising the steps of (a) removing, reducing, depleting, inhibiting, inactivating or capturing AAV-binding antibodies from a blood product obtained from the subject by a process comprising apheresis; and (b) administering a certain amount of recombinant adeno-associated virus (rAAV) vector containing heterologous polynucleotides transcribed into nucleic acids that inhibit, reduce or diminish the expression of the acquisition of function, activity or expression of the protein. 3. The method according to item 1 or 2, wherein the apheresis process includes an AAV-conjugated antibody affinity matrix attached to or immobilized on a substrate. 4. The method according to item 3, wherein the AAV-binding antibody affinity matrix comprises an AAV capsid or AAV capsid fragment attached to or immobilized on a substrate, which binds to the AAV-binding antibody in the blood product. 5. The method according to any one of items 3 to 5, wherein the AAV-binding antibody affinity matrix, immobilized on a substrate, is disposed within a column, apparatus, chamber, device, filter, cartridge, or tube having an inlet and outlet for removing or depleting AAV-binding antibodies from the blood product extracorporeally or in the body when in contact with the AAV-binding antibody affinity matrix. 6. The method according to any one of items 3 to 5, wherein the AAV-binding antibody affinity matrix comprises naturally occurring, unnatural, or synthetic intact AAV empty capsids. 7. The method according to any one of items 3 to 5, wherein the AAV-binding antibody affinity matrix comprises AAV VP1, VP2 and / or VP3 capsid proteins or fragments thereof. 8. The method according to any one of items 3 to 7, wherein the AAV-binding antibody affinity matrix comprises naturally occurring, non-natural, or synthetic AAV VP1, VP2, and / or VP3 capsid proteins. 9. The method according to any one of items 3 to 8, wherein the AAV-conjugated antibody affinity matrix comprises naturally occurring, non-natural, or synthetic AAV VP1, VP2, and / or VP3 capsid protein monomers. 10. The method according to any one of items 3 to 9, wherein the AAV-conjugated antibody affinity matrix comprises naturally occurring, non-natural, or synthetic AAV VP1, VP2, and / or VP3 capsid protein polymers. 11. The method according to any one of items 3 to 10, wherein the AAV-binding antibody affinity matrix comprises AAV VP1, VP2 and / or VP3 capsid proteins having 60% or more sequence identity to naturally occurring, non-natural, or synthetic AAV capsid proteins. 12. The method according to any one of items 3 to 11, wherein the AAV-binding antibody affinity matrix comprises AAV VP1, VP2, and / or VP3 capsid proteins having 60% or more sequence identity with AAV VP1, VP2, and / or VP3 capsid proteins selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1, and SEQ ID NO: 2. 13. The method according to any one of items 3 to 12, wherein the AAV-binding antibody affinity matrix comprises AAV VP1, VP2 and / or VP3 capsid proteins having 60% or more sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. 14. The method according to any one of items 3 to 5, wherein the AAV-binding antibody affinity matrix comprises an anti-idiotype antibody that binds to the AAV-binding antibody in the blood product. 15. The method according to item 14, wherein the anti-idiotype antibody conjugated to the AAV-conjugating antibody conjugates to one or more capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1 and / or SEQ ID NO: 2, or to a derivative or amino acid substitution of one or more capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1 and / or SEQ ID NO: 2. 16. The method according to item 14 or 15, wherein the anti-idiotype antibody that binds to the AAV-conjugated antibody is an antibody fragment. 17. The method according to any one of items 1 to 16, wherein the anti-idiotype antibody conjugated to the AAV-conjugating antibody is IgG, IgA, IgM, IgE, or IgD. 18. The method according to any one of items 14 to 16, wherein the AAV-conjugated antibody affinity matrix is ​​GMP grade. 19. The method according to any one of items 1 to 18, wherein the leaching of the AAV-binding antibody affinity matrix into the blood product obtained from the subject does not substantially harm the subject when the blood product is reintroduced into the subject. 20. The method according to any one of items 1 to 19, wherein the AAV-binding antibody comprises IgG, IgM, IgA, or IgD that binds to the AAV capsid protein. twenty one. The method according to any one of items 1 to 20, wherein the substrate and / or column, apparatus, chamber, device, filter, cartridge, or tube is made of plastic or glass. twenty two. The method according to any one of items 1 to 21, wherein the AAV-conjugated antibody affinity matrix, substrate and / or column, apparatus, chamber, device, filter, cartridge, tube are sterile. twenty three. The method according to any one of items 1 to 22, wherein the amount of AAV-binding antibody present in the blood product prior to the apheresis process is greater than approximately 1:100, in which case 1 part of the blood product is diluted with 100 parts of isotonic buffer to neutralize 50% of the AAV. twenty four. The method according to any one of items 1 to 23, wherein the amount of AAV-binding antibody present in the blood product prior to the apheresis process is greater than approximately 1:1000, in which case a portion of the blood product is diluted with 1000 parts of isotonic buffer, resulting in a 50% neutralization of AAV. twenty five. The method according to any one of items 1 to 24, wherein 20-50%, 50-75%, 75-90%, 90-95%, or 95% or more of the AAV-binding antibody present in the blood product is removed. 26. The method according to any one of items 1 to 25, wherein the AAV-binding antibody present in the blood product after the apheresis process is less than approximately 1:10, in which case 1 part of the blood product is diluted with 10 parts of isotonic buffer to neutralize 50% of the AAV. 27. The method according to any one of items 1 to 26, wherein the AAV-binding antibody present in the blood product after the apheresis process is less than about 1:5, in which case 1 part of the blood product is diluted with 5 parts of isotonic buffer to neutralize 50% of the AAV. 28. The method according to any one of items 1 to 27, wherein the ratio of AAV-binding antibodies present in the blood product after the apheresis process is less than approximately 1:4, in which case 1 part of the blood product is diluted with 4 parts of isotonic buffer to neutralize 50% of the AAV. 29. The method according to any one of items 1 to 27, wherein the ratio of AAV-binding antibodies present in the blood product after the apheresis process is less than approximately 1:3, in which case 1 part of the blood product is diluted with 3 parts of isotonic buffer to neutralize 50% of the AAV. 30. The method according to any one of items 1 to 29, wherein the ratio of AAV-binding antibodies present in the blood product after the apheresis process is less than approximately 1:2, in which case 1 part of the blood product is diluted with 2 parts of isotonic buffer to neutralize 50% of the AAV. 31. The method according to any one of items 1 to 30, wherein the ratio of AAV-binding antibodies present in the blood product after the apheresis process is less than approximately 1:1, in which case a 50% neutralization of AAV is achieved by diluting a portion of the blood product with a portion of isotonic buffer. 32. The method according to any one of items 1 to 31, wherein all or part of the blood product after the method described above is reintroduced or reinjected into the subject. 33. The method according to any one of items 1 to 32, wherein, after step (b), the subject is given a blood product from a donor. 34. The method described in any one of items 1 through 33, wherein step (b) is performed within approximately 72 hours following (a). 35. The method described in any one of items 1 through 33, wherein step (b) is performed within approximately 48 hours following (a). 36. The method described in any one of items 1 to 33, wherein step (b) is performed within approximately 1 to 48 hours after (a). 37. The method described in any one of items 1 through 33, wherein step (b) is performed within approximately 36 hours following (a). 38. The method described in any one of items 1 through 33, wherein step (b) is performed within approximately 24 hours following (a). 39. The method described in any one of items 1 to 33, wherein step (b) is performed within approximately 1 to 24 hours after (a). 40. The method described in any one of items 1 through 33, wherein step (b) is performed within approximately 12 hours following (a). 41. The method described in any one of items 1 through 33, wherein step (b) is performed within approximately 6 hours following (a). 42. The method described in any one of items 1 to 33, wherein step (b) is performed within approximately 3 hours after (a). 43. The method described in any one of items 1 to 33, wherein step (b) is performed within approximately 30 minutes to 6 hours after (a). 44. The method described in any one of items 1 to 33, wherein step (b) is performed within approximately 30 minutes to 3 hours after (a). 45. The method according to any one of items 1 to 44, further comprising, after step (a) but before step (b), analyzing a sample from the subject for the amount of AAV-binding antibody present in the sample. 46. The method according to any one of items 1 to 45, further comprising, after step (b), analyzing a sample from the subject for the amount of AAV-binding antibody present in the sample. 47. The method according to item 45 or 46, wherein the sample analyzed from the subject is a blood product. 48. The method according to any one of items 1 to 47, wherein the blood product is plasma. 49. The method according to any one of items 1 to 48, wherein the subject 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, lysosomal storage disorder, copper or iron storage disorder (e.g., Wilson's disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative disorder, cancer, type 1 or type 2 diabetes, Gaucher disease, Hurler's disease, adenosine deaminase deficiency, a metabolic disorder (e.g., glycogen storage disorder), a retinal degenerative disease (e.g., RPE65 deficiency, colloideremia, and other eye diseases), a solid organ disease (e.g., brain, liver, kidney, heart), or an infectious virus (e.g., hepatitis B and C, HIV, etc.), a bacterial or fungal disease. 50. The method according to any one of items 1 to 49, wherein the disease is caused by the loss or reduction of expression of the gene encoding the protein. 51. The method according to any one of items 1 to 50, wherein the disease is a blood coagulation disorder. 52. The method according to any one of items 1 to 51, wherein the disease is hemophilia A, hemophilia A patient with inhibitory antibodies, hemophilia B, any coagulation factor: VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor deficiency, or FV / FVIII combined deficiency, or thalassemia, vitamin K epoxydoreductase C1 deficiency, or gamma carboxylase deficiency. 53. The method according to any one of items 1 to 50, wherein the disease is anemia, trauma-related bleeding, injury, thrombosis, thrombocytopenia, stroke, coagulation disorders, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors); and platelet disorders, such as Bernard-Soulier syndrome, Glanzmann thromblastemia, or storage pool deficiency. 54. The method described in any one of items 1 to 50, wherein the disease affects or originates from the central nervous system (CNS). 55. The method according to any one of items 1 to 50, wherein the disease is a neurodegenerative disease. 56. The method according to item 54 or 55, wherein the CNS or neurodegenerative disease is Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, polyglutamine repeat disorder, or Parkinson's disease. 57. The method according to item 55 or 56, wherein the CNS or neurodegenerative disease is a polyglutamine repeat disease. 58. The method according to item 57, wherein the polyglutamine repeat disorder is spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17). 59. The aforementioned heterogeneous polynucleotides include 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), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor α (TGFα), and platelet-derived growth factor (P). The method according to item 1 or any one of items 3 to 58, encoding a protein selected from the group consisting of DGF), insulin growth factor I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neuruturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase. 60. The method according to item 1 or any one of items 3 to 58, wherein the heterogeneous polynucleotide encodes a protein selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-17), monocyte chemotactic proteins, leukemia suppressor factors, granulocyte-macrophage colony-stimulating factors, Fas ligands, tumor necrosis factor α and β, interferon α, β and γ, stem cell factors, flk-2 / flt3 ligands, 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, and class I and class II MHC molecules. 61. The aforementioned heterogeneous polynucleotides are involved in the acquisition of functional blood coagulation factors such as CFTR (cystic fibrosis transmembrane regulatory protein), blood coagulation factors (factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), antibodies, retinal pigment epithelium-specific 65kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), and metal transporters (ATP). 7A or ATP7), sulfamidase, enzymes involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factors 3 and 4, brain-derived neurotrophic factor, glial growth factor, transforming growth factors α and β, cytokines, α-interferon, β-interferon, interferon-γ, 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 adenomatous polyposis (APC)), peptides with immunomodulatory properties, Tolerogenic or immunogenic peptides or protein T regitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (colloideremia), LCA5 (LCA-level cylin), ornithine keto acid aminotransferase (rotational atrophy), retinosuxin 1 (X-linked retinal detachment), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 26 hearing loss),The methods described in item 1 or any one of items 3-58, which encode gene products involved in ACHM2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, lysosomal storage disorders (e.g., 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 donor sequences used as repair templates for genome editing). 62. The method according to any one of items 2 to 58, wherein the heterogeneous polynucleotide encodes an inhibitory nucleic acid. 63. The method according to any one of items 2 to 58, wherein the inhibitory nucleic acid is selected from the group consisting of siRNA, antisense molecules, miRNA, RNAi, ribozymes, and shRNA. 64. The inhibitory nucleic acid is a pathogenic gene, a transcript of a pathogenic gene, or a transcript of a gene associated with polynucleotide repeat disease, the huntingtin (HTT) gene, a gene associated with dentatorubropallidolusyan atropy (atropin 1, ATN1), the androgen receptor on the X chromosome in spinal and bulbar muscular atrophy, and Ca, encoded by human attaxin-1, -2, -3, and -7 (CACNA1A). v2.1 P / Q voltage-gated calcium channel, TATA-binding protein, also known as ATXN8OS, ataxin 8 reverse chain, serine / threonine protein phosphatase 2A 55kDa regulatory subunit B beta isoform (1, 2, 3, 6, 7, 8, 12) in spinocerebellar ataxia. FMR1 (Fragile X Intellectual Disability 1) in Fragile X Syndrome, FMR1 (Fragile X Intellectual Disability 1) in Fragile X-associated Tremor / Ataxia Syndrome, FMR1 (Fragile X Intellectual Disability 2) or AF4 / FMR2 family member 2 in Fragile XE Intellectual Disability; Myotonin-protein kinase (MT-PK) in Myotonic Dystrophy; Frataxin in Friedreich's Ataxia; Mutants of the superoxide dismutase 1 (SOD1) gene in Amyotrophic Lateral Sclerosis; Genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; Apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercoloesterolemia; HIV Tat in HIV infection, human immunodeficiency virus transactivator of transcription genes; HIV TAR in HIV infection, HIV TAR, human immunodeficiency virus transactivator response element gene; CC chemokine receptor (CCR5) in HIV infection; Roussarcoma virus (RSV) nucleocapsid protein in RSV infection; liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function kidney graft or kidney injury; protein kinase N3 (PKN3) in progressive, recurrent, or metastatic solid malignancies; LMP2, also known as proteasome subunit beta-9 (PSMB9), metastatic melanoma; LMP7, also known as proteasome subunit beta-8 (PSMB8), metastatic melanoma; MECL1, also known as proteasome subunit beta-10 (PSMB10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors; apoptosis-suppressing B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia;Ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors; diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection; beta-catenin in familial adenomatous polyposis; beta-2 adrenergic receptor; glaucoma; RTP801 / Redd1, also known as DAN 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 neivascularization; caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6A in congenital onychoplasty N17K mutant protein; influenza A virus genome / gene sequence in influenza infection; SARS coronavirus genome / gene sequence in severe acute respiratory syndrome (SARS) infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola filovirus genome / gene sequence in Ebola infection; hepatitis B and C virus genome / gene sequence in hepatitis B and C infection; herpes simplex virus (HSV) genome / gene sequence in herpes simplex virus (HSV) infection, coxsackievirus B3 genome / gene sequence in coxsackievirus B3 infection; silencing of pathogenic alleles of genes such as torsin A (TOR1A) in primary dystonia (allele-specific silencing), pan-class I and HLA allele-specific in transplantation; or binding to mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP), as described in item 63. 65. The method according to any one of items 1 to 64, wherein the heterogeneous polynucleotide encodes a gene editing nuclease. 66. The method according to item 65, wherein the gene editing nuclease comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). 67. The method according to any one of items 1 to 64, wherein the heterologous polynucleotide encodes a functional type II CRISPR-Cas9; and / or a guide RNA sequence; and / or a donor nucleic acid sequence for modification or substitution of a target gene. 68. The method according to any one of items 1 to 67, wherein step (a) and / or step (b) are performed two or more times. 69. The method described in any one of items 1 to 68, wherein the subject is a human. 70. The AAV-binding antibody affinity matrix is ​​immobilized on a substrate disposed within a column, apparatus, chamber, device, filter, cartridge, or tube, and has an inlet and outlet for removing or depleting AAV-binding antibodies from a blood product extracorporeally or intracorporeally when in contact with the AAV-binding antibody affinity matrix. 71. An AAV-conjugated antibody affinity matrix as described in item 70, comprising naturally occurring, non-natural, or synthetic intact AAV empty capsids. 72. An AAV-binding antibody affinity matrix as described in item 70, comprising AAV VP1, VP2, and / or VP3 capsid proteins or fragments thereof. 73. The AAV-binding antibody affinity matrix according to item 70, wherein the AAV-binding antibody affinity matrix comprises naturally occurring, non-natural, or synthetic AAV VP1, VP2, and / or VP3 capsid proteins. 74. The AAV-binding antibody affinity matrix according to item 70, wherein the AAV-binding antibody affinity matrix comprises naturally occurring, non-natural, or synthetic AAV VP1, VP2, and / or VP3 capsid protein monomers. 75. The AAV-conjugated antibody affinity matrix according to item 70, wherein the AAV-conjugated antibody affinity matrix comprises naturally occurring, non-natural, or synthetic AAV VP1, VP2, and / or VP3 capsid protein polymers. 76. The AAV-binding antibody affinity matrix according to item 70, wherein the AAV-binding antibody affinity matrix comprises AAV VP1, VP2 and / or VP3 capsid proteins having 60% or more sequence identity to naturally occurring, non-natural, or synthetic AAV capsid proteins. 77. The AAV-binding antibody affinity matrix according to item 70, wherein the AAV-binding antibody affinity matrix comprises AAV VP1, VP2, and / or VP3 capsid proteins having 60% or more sequence identity with AAV VP1, VP2, and / or VP3 capsid proteins selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1, and SEQ ID NO: 2. 78. The AAV-binding antibody affinity matrix according to item 70, wherein the AAV-binding antibody affinity matrix comprises AAV VP1, VP2 and / or VP3 capsid proteins having 60% or more sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. 79. The AAV-binding antibody affinity matrix according to item 70, wherein the AAV-binding antibody affinity matrix comprises an anti-idiotype antibody that binds to the AAV-binding antibody in the blood product. 80. The AAV-binding antibody affinity matrix according to item 79, wherein the anti-idiotype antibody that binds to the AAV-binding antibody binds to one or more capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1 and / or SEQ ID NO: 2, or a derivative or amino acid substitution of one or more capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1 and / or SEQ ID NO: 2. 81. The AAV-conjugated antibody affinity matrix according to item 79, wherein the anti-idiotype antibody that binds to the AAV-conjugated antibody is an antibody fragment. 82. The AAV-binding antibody affinity matrix according to item 79, wherein the anti-idiotype antibody that binds to the AAV-binding antibody is IgG, IgA, IgM, IgE, or IgD. 83. The AAV-binding antibody affinity matrix described in any one of items 70 to 82, wherein the AAV-binding antibody affinity matrix is ​​GMP grade. 84. The AAV-binding antibody affinity matrix according to any one of items 70 to 82, wherein the leaching of the AAV-binding antibody affinity matrix into a blood product obtained from a subject does not substantially harm the subject when the blood product is reintroduced into the subject. 85. An AAV-binding antibody affinity matrix according to any one of items 70 to 82, wherein the AAV-binding antibody comprises IgG, IgM, IgA, or IgD that binds to the AAV capsid protein. 86. The AAV-conjugated antibody affinity matrix according to any one of items 70 to 82, wherein the substrate and / or column, apparatus, chamber, device, filter, cartridge, or tube is made of plastic or glass. 87. The AAV-conjugated antibody affinity matrix according to any one of items 70 to 82, wherein the AAV-conjugated antibody affinity matrix, substrate and / or column, apparatus, chamber, device, filter, cartridge, or tube is sterile.

[0161] [Related applications]

[0001] This patent application claims priority to U.S. Patent Application No. 62 / 533,579, filed on 17 July 2017. The entirety of the said application, including all text, tables, drawings and arrangements, is incorporated herein by reference.

Claims

1. 1. A composition comprising a recombinant adeno-associated virus (rAAV) vector for delivery of a heterologous polynucleotide to a human subject in need of treatment for a disease caused by loss of function or activity of a protein or peptide, comprising: the rAAV vector comprises: (A) the heterologous polynucleotide, wherein the heterologous polynucleotide encodes a protein or peptide that provides or supports the function or activity; and (B) a capsid; The composition comprises: (i) removing a pre-apheresis blood product from said subject; (ii) passing the pre-apheresis blood product through an AAV-binding antibody affinity matrix comprising the capsid to produce a post-apheresis blood product having a reduced amount of antibody binding to the rAAV vector compared to the pre-apheresis blood product, wherein the capsid is an empty capsid that does not contain the heterologous polynucleotide, and the capsid comprises: (a) is non-naturally occurring, synthetic, or has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, and Rh74; or (b) comprising VP1 of SEQ ID NO: 1 or VP1 of SEQ ID NO: 2; (iii) infusing the post-apheresis blood product into the subject; and (iv) administering the rAAV vector to the subject, the composition being intended for administration to the subject undergoing apheresis and rAAV vector administration.

2. 1. A composition comprising a recombinant adeno-associated virus (rAAV) vector for delivery of a heterologous polynucleotide to a human subject in need of treatment for a disease caused by gain of protein function, activity, or expression, comprising: the rAAV vector comprises: (A) the heterologous polynucleotide, which is transcribed into a nucleic acid that reduces expression of the protein; and (B) a capsid; The composition comprises: (i) removing a pre-apheresis blood product from said subject; (ii) passing the pre-apheresis blood product through an AAV-binding antibody affinity matrix comprising the capsid to produce a post-apheresis blood product having a reduced amount of antibody binding to the rAAV vector compared to the pre-apheresis blood product, wherein the capsid is an empty capsid that does not contain the heterologous polynucleotide, and the capsid comprises: (a) is non-naturally occurring, synthetic, or has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, and Rh74; or (b) comprising VP1 of SEQ ID NO: 1 or VP1 of SEQ ID NO: 2; (iii) infusing the post-apheresis blood product into the subject; and (iv) administering the rAAV vector to the subject, the composition being intended to be administered to the subject undergoing apheresis and rAAV vector administration.

3. (i) the subject has an rAAV vector antibody titer of up to 1:1000 prior to the apheresis, and step (iv) is within 1 hour of step (iii); (ii) the subject has an rAAV vector antibody titer of up to 1:300 prior to the apheresis, and step (iv) is within 3 hours of step (iii). (iii) the subject has an rAAV vector antibody titer of up to 1:100 prior to the apheresis, and step (iv) is within 12 hours of step (iii); or (iv) the subject has an rAAV vector antibody titer of up to 1:30 prior to the apheresis, and step (iv) is within 24 hours of step (iii).

4. 3. The composition of claim 1 or 2, wherein the bound antibody affinity matrix is ​​immobilized on a substrate and disposed within a column, apparatus, chamber, device, filter, cartridge, or tube, the column, apparatus, chamber, device, filter, cartridge, or tube having an inlet and an outlet for in vitro or in vivo removal or depletion of AAV-binding antibodies from the blood product by contact with the AAV-binding antibody affinity matrix.

5. 3. The composition of claim 1, wherein the empty capsid is linked to either CNBr-activated Sepharose 4, activated thiol Sepharose 4B, carbodiimide-based linkage of the carboxyl group to Sepharose 4B via an 11-atom hydrophilic spacer arm, or epoxy-activated Sepharose 6B.

6. 3. The composition of claim 1 or 2, wherein the AAV-binding antibody affinity matrix comprises a capsid protein selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, and Rh74.

7. 3. The composition of claim 1 or 2, wherein the empty capsid comprises AAV VP1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

8. 3. The composition of claim 1 or 2, wherein the AAV-binding antibody affinity matrix, substrate, column, apparatus, chamber, device, filter, cartridge, and tube are sterile.

9. The composition of claim 1 or 2, wherein the blood product is plasma.

10. The composition of claim 1 or 2, wherein the disease is a blood clotting disorder.

11. 3. The composition according to claim 1, wherein the disease is hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, a deficiency of any of coagulation 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.

12. 3. The composition of claim 1 or 2, wherein the disease is Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, polyglutamine repeat disease, or Parkinson's disease.

13. The composition of claim 12, wherein the disease is a polyglutamine repeat disease.

14. 14. The composition of claim 13, wherein the polyglutamine repeat disease is a spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

15. The composition of claim 2 , wherein the heterologous polynucleotide encodes an inhibitory nucleic acid.

16. The composition of claim 15, wherein the inhibitory nucleic acid is selected from the group consisting of siRNA, antisense molecules, miRNA, ribozymes and shRNA.

17. 3. The composition of claim 1 or 2, wherein step (ii) is carried out two or more times.

18. 3. The composition of claim 1 or 2, wherein the subject has an rAAV vector antibody titer of about 1:1000 prior to the apheresis, and step (iv) is about 1 hour after step (iii).

19. 3. The composition of claim 1 or 2, wherein the subject has an rAAV vector antibody titer of about 1:300 prior to the apheresis, and step (iv) is about 3 hours after step (iii).

20. 3. The composition of claim 1 or 2, wherein the subject has an rAAV vector antibody titer of about 1:100 prior to the apheresis, and wherein step (iv) is about 12 hours after step (iii).

21. 3. The composition of claim 1 or 2, wherein the subject has an rAAV vector antibody titer of about 1:30 prior to the apheresis, and wherein step (iv) is about 24 hours after step (iii).