WO2023036054A2

WO2023036054A2 - Composition and method for treating hemophilia

Info

Publication number: WO2023036054A2
Application number: PCT/CN2022/116702
Authority: WO
Inventors: Zhongdong SHI
Original assignee: Inspirar Limited
Priority date: 2021-09-08
Filing date: 2022-09-02
Publication date: 2023-03-16
Also published as: WO2023036054A3

Abstract

The present invention relates to a method for treating hemophilia A the compositions and the use of compositions for the treatment of hemophilia A.

Description

COMPOSITION AND METHOD FOR TREATING HEMOPHILIA

CROSS REFERNCE

This application claims the benefit of priority to PCT Application No. PCT/CN2021/117166, filed on September 8, 2021, which is herein expressly incorporated by reference in its entirety, including any drawings.

BACKGROUND

Hemophilia A and B are congenital bleeding disorders caused by a deficiency or complete absence of coagulation factor VIII (FVIII) or factor IX (FIX) , respectively. These X-linked disorders represent the most inherited deficiencies of clotting factors, occurring in approximately one per 5000 and one per 50,000 male births, with no racial predilection. Type A, the most common type, is caused by a deficiency of factor VIII, one of the proteins that helps blood to form clots. This type is known as classic hemophilia.

Although hemophilia is usually diagnosed at birth, the disorder can also be acquired later in life if the body begins to produce antibodies that attack and destroy clotting factors. The development of inhibitory alloantibodies to FVIII can severely complicate the treatment of genetic cases. Rarely, development of autoantibodies to FVIII results in acquired hemophilia A.

The symptom of hemophilia is bleeding and depends on whether the hemophilia is the mild, moderate, or severe form of the disorder. For severe hemophilia, unprovoked (spontaneous) bleeding episodes occur often; for moderate hemophilia, prolonged bleeding tends to occur after a more significant injury; for mild hemophilia, a patient might have unusual bleeding, but only after a major injury, surgery, or trauma. Nonetheless, people with hemophilia may have any type of internal bleeding, but most often it occurs in the muscles and joints, such as the elbows, knees, hips, shoulders, and ankles. Thus, it is important to identify efficient and effective treatment for hemophilia A and hemophilia B.

SUMMARY

Currently, there is a need for the development of drugs and methods that can be effective for treating hemophilia A. The compositions and methods disclosed herein solve the above-mentioned problems and meet the need.

In one aspect, the present disclosure provides a polynucleotide comprising a nucleic acid encoding a human Factor VIII peptide, wherein the polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 5-22; wherein the Factor VIII peptide lacks a B domain.

In another aspect, the present disclosure provides a polynucleotide comprising a nucleic acid encoding a human Factor VIII (FVIII) peptide, wherein the polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 22; wherein the Factor VIII peptide lacks a B domain.

In some embodiments, the polynucleotide comprises a sequence of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some embodiments, the polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to SEQ ID NOs: 6, 10, 18, or 20. In some embodiments, the polynucleotide comprises a sequence of SEQ ID NOs: 6, 10, 18, or 20.

In some embodiments, the polynucleotide is a recombinant polynucleotide. In some embodiments, the polynucleotide is a synthetic polynucleotide. In some embodiments, the polynucleotide is a combination of a recombinant polynucleotide and a synthetic polynucleotide. In some embodiments, the polynucleotide is codon optimized. In some embodiments, the nucleic acid encoding the human Factor VIII peptide comprises a reduced number of CpG dinucleotides as compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain. In some embodiments, the nucleic acid encoding the human Factor VIII peptide comprises less than about 5 CpG dinucleotides. In some embodiments, the nucleic acid encoding the human Factor VIII peptide does not comprise a CpG dinucleotide.

In some embodiments, the nucleic acid encoding the human Factor VIII peptide has a reduced immunogenicity compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain. In some embodiments, an expression level of the human Factor VIII peptide is increased by at least about 10%compared to an expression level of a wild-type Factor VIII peptide without a B domain encoded by a wild-type nucleic acid. In some embodiments, the increased expression level of said human Factor VIII peptide activates a less unfolded protein response (UPR) by at least 5%compared to an expression level of a wild-type Factor VIII peptide without a B domain encoded by a wild-type nucleic acid. In some embodiments, the increased expression level of the human Factor VIII peptide reduces an endoplasmic reticulum (ER) stress response by at least 5%compared to an expression level of a wild-type Factor VIII peptide without a B domain encoded by a wild-type nucleic acid. In some embodiments, the polynucleotide comprises a promoter operably connected to the nucleic acid encoding the human Factor VIII peptide.

In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the liver-specific promoter is a transthyretin (TTR) promoter. In some embodiments, the polynucleotide is deoxyribonucleic acid (DNA) . In some embodiments, the polynucleotide is ribonucleic acid (RNA) .

In another aspect, the present disclosure provides a polynucleotide comprising a nucleic acid encoding a human Factor VIII peptide, wherein said polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, and 22; wherein said Factor VIII peptide lacks a B domain.

In some embodiments, the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, and 22. In some embodiments, the polynucleotide comprises a sequence with at least about 90%sequence identity to SEQ ID NOs: 17, 18, 19, 20, 21, or 22. In some embodiments, the polynucleotide comprises a sequence of SEQ ID NOs: 17, 18, 19, 20, 21, or 22.

In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) particle, comprising a polynucleotide comprising a nucleic acid encoding a human Factor VIII peptide lacking a B domain; wherein the Factor VIII peptide comprises a F309S mutation; and wherein said rAAV particle encodes an AAV capsid protein and wherein said AAV capsid protein is selected from the group consisting of AAV2, AAV5, AAV6, AAV8, AAV9, AAVHSC, AAV3B, AAVDJ, LK03, NP59, and KP1.

In some embodiments, the rAAV particle is a single-stranded AAV (ssAAV) vector. In some embodiments, the AAV capsid protein is AAV5.

In some embodiments, the AAV capsid protein is AAV2. In some embodiments, the AAV capsid protein is AAV3B. In some embodiments, the AAV capsid protein is AAV6. In some embodiments, the AAV capsid protein is AAV8. In some embodiments, the AAV capsid protein is AAV9. In some embodiments, the AAV capsid protein is AAVHSC. In some embodiments, the AAV capsid protein is AAV-DJ. In some embodiments, the AAV capsid protein is LK03. In some embodiments, the AAV capsid protein is NP59. In some embodiments, the AAV capsid protein is KP1.

In some embodiments, the polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identify to SEQ ID NOs: 17, 18, 19, 20, 21, or 22. In some embodiments, the polynucleotide comprises a sequence of SEQ ID NOs: 17, 18, 19, 20, 21, or 22.

In some embodiments, the polynucleotide is codon optimized. In some embodiments, the nucleic acid encoding the human Factor VIII peptide comprises less than about 5 CpG dinucleotides. In some embodiments, the nucleic acid encoding the human Factor VIII peptide does not comprise a CpG dinucleotide.

In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) particle, prepared by transfecting the composition of the nucleic acid disclosed herein into a host cell. In some embodiments, the host cell is an insect cell, a human cell, or an animal cell. In some embodiments, the insect cell is a Drosophila S2 cell or a Sf9 cell. In some embodiments, the animal cell is a fibroblast, a Chinese hamster ovary (CHO) cell, a COS cell, a murine myeloma (NS0) cell, a HeLa cell, or a Baby Hamster Kidney (BHK) cell. In some embodiments, the human cell is a human embryonic kidney 293 (HEK293) cell, a HEK293 derivative (such as 293T) , a human fibrosarcoma (HT-1080) cell, a differentiated hepatocyte-derived carcinoma (Huh-7) cell, or a PER. C6 cell.

In another aspect, the present disclosure provides a pharmaceutical composition, comprising (a) a recombinant nucleic acid disclosure herein or a recombinant adeno-associated virus (rAAV) particle disclosed herein as well; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises serum free media, a lipid, a nanoparticle, vitamin K, surfactant, phosphate buffered saline (PBS) . In some embodiments, the surfactant is Pluronic acid F-68.

In another aspect, the present disclosure provides methods for treating hemophilia A, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to a subject in need thereof. In some embodiments, the pharmaceutical composition is administered via intravenous injection. In some embodiments, the pharmaceutical composition is administered at a dose of about 10 ⁸ vg/kg -10 ¹⁵ vg/kg.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “FIG. ” herein) , of which:

FIG. 1 illustrates the expression levels of B domain-deleted (BDD) human FVIII factor variant and mutant peptides encoded by various nucleic acids disclosed herein after transfection in Huh-7 cells.

FIG. 2 illustrates the activities of the BDD human FVIII factor variant and mutant peptides encoded by various nucleic acids from FIG. 1 in cell culture supernatants after transfection.

FIG. 3 illustrates the specific activities of the BDD human FVIII factor variant and mutant peptides encoded by different nucleic acids from FIG. 1 in cell culture supernatants after transfection.

FIG. 4 illustrates human FVIII concentrations in FVIII-KO mouse plasma samples on day 14 post-dosing of various AAV vectors comprising the BDD human FVIII factor variant and mutant peptides encoded by different nucleic acids as disclosed herein.

FIG. 5 illustrates human FVIII activities in FVIII-KO mouse plasma samples on day 14 post-dosing of various AAV vectors comprising the BDD human FVIII factor variant and mutant peptides encoded by different nucleic acids as disclosed herein.

FIG. 6 illustrates human FVIII concentrations in FVIII-KO mouse plasma samples on day 30 post-dosing of various AAV vectors comprising the BDD human FVIII factor variant and mutant peptides encoded by different nucleic acids as disclosed herein.

FIG. 7 illustrates human FVIII activities in FVIII-KO mouse plasma samples on day 30 post-dosing of various AAV vectors comprising the BDD human FVIII factor variant and mutant peptides encoded by different nucleic acids as disclosed herein.

DETAILED DESCRIPTION

Although described herein have been shown and described various embodiments of the present invention, but for obvious to the person skilled readily apparent that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art would recognize many variations, changes, and substitutions. It should be understood that the present invention may be employed herein, according to various alternatives to the next embodiment.

Unless otherwise stated, the practice of some embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012) ; the series Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds. ) ; the series Methods In Enzymology (Academic Press, Inc. ) , PC 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995) ) , Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications , 6th Edition (RI Freshney, ed. (2010) ) .

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods, and materials are described below. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

DEFINITION

As used in the specification and claims, the singular forms "a" , "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "rAAV particle" includes one or more rAAV particles.

The term "about" or "approximately" refers to within the acceptable error range of a specific value determined by a person of ordinary skill in the art, which will depend in part on how the value is measured or determined, that is, the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20 %, up to 10 %, up to 5 %, or up to 1 %of a given value. Or, particularly for biological systems or processes, the term may represent an order of magnitude of the value, preferably within 5 times, more preferably within 2 times. Where specific values are described in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within the acceptable error range of the specific value.

It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. The terms “and/or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C. ” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

As used in this specification and claim (s) , the words “comprising” (and any form of comprising, such as “comprise” and “comprises” ) , “having” (and any form of having, such as “have” and “has” ) , “including” (and any form of including, such as “includes” and “include” ) or “containing” (and any form of containing, such as “contains” and “contain” ) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

As used herein, “some embodiments, ” “an embodiment, ” “one embodiment, ” “embodiments” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

As used herein, the terms "polypeptide, " "peptide, " and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polypeptide can be linear, cyclic or branched. The polypeptide can contain modified amino acids. The polypeptide can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified, such as by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic treatment, phosphorylation, prenylation, racemization, selenization, transfer RNA-mediated addition of amino acids to proteins (such as arginylation) , ubiquitination, or any other operations, such as conjugation with labeling components. As used herein, the term "amino acid" refers to natural and /or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived" from a given protein refers to the origin of the polypeptide. Preferably, the polypeptide has an amino acid sequence that is substantially the same as the amino acid sequence of the polypeptide encoded in the sequence, or a part thereof, wherein the part consists of at least 10-20 amino acids or at least 20-30 amino acids or at least 30-50 amino acids. Alternatively, the polypeptide can be identified immunologically with the polypeptide encoded in the sequence. The term also includes polypeptides expressed from a designated nucleic acid sequence. As used herein, the term "domain" refers to a part of a protein that is physically or functionally distinguished from other parts of the protein or peptide. Physically defined domains include amino acid sequences that are extremely hydrophobic or hydrophilic, such as those that are membrane-bound or cytoplasmic-bound. The domain can also be defined by internal homology caused by gene duplication, for example. Functionally defined domains have different biological functions. For example, the antigen-binding domain refers to the part of the antigen-binding unit or antibody that binds to the antigen. The functionally defined domain does not need to be encoded by a continuous amino acid sequence, and the functionally defined domain may contain one or more physically defined domains.

As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including but not limited to D or L optical isomers, as well as amino acid analogs and peptidomimetics. Standard one-letter or three-letter codes are used to designate amino acids. In this context, amino acids are usually represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, in the case of a polypeptide, a "sequence" is the sequence of amino acids in the polypeptide in the direction from the amino terminal to the carboxy terminal, wherein the residues adjacent to each other in the sequence are continuous in the primary structure in the polypeptide. The sequence can also be a linear sequence of a part of a polypeptide known to contain additional residues in one or two directions.

As used herein, "identity" , "homology" or "sequence identity" refers to the similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using programs such as Emboss Needle or BestFit to determine the sequence identity, similarity or homology between two different amino acid sequences, the default settings can be used, or an appropriate scoring matrix can be selected, such as blosum45 or blosum80, to optimize identity, similarity, or homology score. Preferably, homologous polynucleotides are those that hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99%sequence identity compared to these polynucleotides. When a sequence of comparable length optimally aligned, homologous polypeptide preferably has at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%sequence identity, or with a at least 99%sequence identity.

With regard to the antigen-binding unit determined herein, the "percentage of sequence identity (%) " is defined as after aligning the sequences and introducing gaps if necessary to obtain the maximum sequence identity percentage, and not removing any conservative substitutions are regarded as part of sequence identity, and the percentage of amino acid residues in the query sequence that are identical to the amino acid residues of the second, reference polypeptide sequence or part thereof. The alignment aimed at determining the percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, such as using publicly available computer software, such as BLAST, BLAST-2 (preferred) , ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment over the full length of the sequence being compared. The percent identity can be measured over the length of the entire defined polypeptide sequence, or can be measured over a shorter length, for example, the length of a fragment taken from a larger, defined polypeptide sequence, such as A fragment of at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, or at least 200 consecutive residues. These lengths are exemplary only, and it should be understood that the forms herein shown in the drawings, or the sequence supported in the Sequence Listing can be used to describe any fragment length thereon may be measured with a percentage of the length.

The proteins described herein may have one or more modifications relative to the reference sequence. The modification may be deletion, insertion or addition, or substitution or substitution of amino acid residues. "Deletion" refers to a change in amino acid sequence due to the lack of one or more amino acid residues. "Insert" or "Add" means results in the addition of one or more as compared to the reference sequence amino acid residues of amino acid sequence changes. "Substitution" or "substitution" refers to the replacement of one or more amino acids with different amino acids. Herein, the mutation of the antigen-binding unit relative to the reference sequence can be determined by comparing the antigen-binding unit with the reference sequence. The optimal alignment of sequences for comparison can be performed according to any known method in the art.

As used herein, the term "sub isolated" refers to cells and other aspects of components of the separation surface, wherein in nature, polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof It is associated with it under normal circumstances. Those skilled in the art know that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be "isolated" to distinguish them from their naturally occurring counterparts. In addition, "concentrated" , "isolated" or "diluted" polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are distinguishable from their naturally occurring counterparts because of the concentration or number of molecules per unit volume Greater than ( "concentrated" ) or less than from its naturally occurring counterpart ( "isolated" ) . Enrichment can be measured based on absolute amounts, such as the weight of solution per unit volume, or it can be measured relative to the second, potentially interfering species present in the source mixture.

The terms "polynucleotide" , "nucleic acid" , "nucleotide" and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length (whether deoxyribonucleotides or ribonucleotides) or their analogs. The polynucleotide may have any three-dimensional structure, and may perform any known or known functions. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci determined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomes an RNA, ribozymes, the cDNA, recombinant polynucleotides, branched polynucleotides, isolated plasmids, vectors, any sequence DNA isolated, any sequence of the RNA, nucleic acid probes, primers, or a synthetic oligonucleotide DNA. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component.

When applied to polynucleotides, "recombinant" means that the polynucleotide is cloned, restriction digestion and/or ligation steps, and other procedures that produce constructs different from those found in nature.

The terms "gene" or "gene fragment" are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame, the open reading frame capable of encoding a particular protein after transcription and translation. Gene or gene fragment may be a gene group, the cDNA, or synthetic, as long as the polynuclear nucleotide comprises at least one open reading frame, which may cover the entire open reading frame coding region or a section thereof.

The term "operably linked" or "operably linked" refers to a juxtaposition, wherein the relationship in which the components so described permitting them to their intended function in a manner. For example, if a promoter sequence promotes transcription of a coding sequence, the promoter sequence is operably linked to the coding sequence.

As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA, and/or the process by which transcribed mRNA (also referred to as "transcripts" ) is subsequently translated into peptides, polypeptides or proteins. The transcripts and the encoded polypeptides are collectively referred to as group gene product. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. Carrier may be through transformation, transduction or transfection into a host cell, so that the carried genetic material is expressed in a host cell. Vectors are well known to those skilled in the art, including but not limited to, plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC) , bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage body and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses) , adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus) , poxviruses, baculoviruses, papillomaviruses, and papillae Polyoma vacuole virus (such as SV40) . A vector may contain a variety of expression control elements, including but not limited to, a promoter sequence column, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may also contain an origin of replication site.

The term "codon optimization" as used herein refers to the use of redundancy in the genetic code to change the nucleotide sequence while maintaining the same protein sequence it encodes. In some cases, codon optimization may be increased or decreased to promote the expression of the protein encoded, this is done by for a particular cell type of codon usage preferences as tRNA in the cell types of the relative abundance, so that the nucleotide sequences comprising the codons are adapted to this preference. In some cases, expression can also be reduced by selecting codons for tRNAs that are known to be rare in specific cell types. In some cases, by-optimized code can also be increased sequence replication of fidelity, i.e., in a multi-period polynucleotide replication cycle such as cloning in less mutation.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, such as yeast cells or Aspergillus, etc. Fungal cells, such as insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

An "effective amount" as used herein refers to at least the minimum amount required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary with the patient's disease state, age, sex, weight and other factors. An effective amount is also an amount in which the therapeutic benefit exceeds any toxic or adverse effects of the treatment. In the treatment of cancer or tumor, the effective dose of the drug can have the following effects: reduce the number of cancer cells, reduce tumor size, inhibit the infiltration of cancer cells into peripheral organs, inhibit tumor metastasis, inhibit tumor growth to a certain extent and/or to a certain extent alleviate one or more symptoms related to the disease. The effective amount can be administered in one or more applications.

As used herein, the terms "recipient" , "individual" , "subject" , "host" , and "patient" are used interchangeably herein, and refer to those any mammalian subject that wish to be diagnosed or treated, especially human.

As used herein, the terms "treatment" and "treatment" refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and/or may be therapeutic in terms of partially or completely stabilizing or curing the disease and/or adverse reactions attributed to the disease. "Treatment" as used herein encompasses any treatment of diseases in mammals, such as mice, rats, rabbits, pigs, primates, including humans and other apes, especially humans, and the term includes: (a) preventing a disease or symptom from occurring in subjects who may be susceptible to the disease or symptom but not yet diagnosed; (b) inhibiting disease symptoms; (c) preventing the development of the disease; (d) relieving symptoms of the disease; (e) causing the disease or symptoms to subside; or any combination thereof. The term "kit" as used herein refers to a combination packaged for common use or commercially available. For example, the kit of the present disclosure may include the composition of the present disclosure, and instructions for using the composition or the kit. The term "instructions" refers to the explanatory inserts usually contained in commercial packages of therapeutic products, which contain information about indications, use, dosage, administration, combination therapy, contraindications and/or warnings about the use of such therapeutic products.

As used herein, the term “Factor VIII” generally refers to all wild-type and variant Factor VIII sequences, for example, a wild-type Factor VIII sequence without a B domain.

HEMOPHILIA A

The present disclosure presents a gene therapy that may be used to treat a subject suffering or at risk of suffering hemophilia A. As discussed before, hemophilia A is a rare, inherited bleeding disorder in which blood cannot clot normally at the site of a wound or injury. Hemophilia A is the most common type and is caused by a deficiency of Factor VIII (FVIII) peptide.

FVIII is a glycoprotein cofactor that serves as a critical component in the intrinsic blood coagulation pathway. FVIII is a large multidomain glycoprotein with domain structure A1-A2-B-A3-C1-C2, wherein the heavy chain is composed of domains A1-A2-B, and the light chain of domains A3-C1-C2. The A domains are bordered by short spacers that contain clusters of aspartic acid and glutamic acid residues, called acidic regions. The large B domain is encoded by a single large exon and has no detectable homology to any other known genes. It is extensively glycosylated on asparagine, serine, and threonine residues. The C domains occur twice in the carboxy terminus of the FVIII light chain and exhibit homology to proteins that bind glycoconjugates and negatively charged phospholipids. FVIII, a divalent metal ion-dependent heterodimer, contains a single copper atom but, the role of this metal in the structure and function of the cofactor is unclear. Furthermore, association of FVIII heavy chain (A1-A2-B domains) and light chain (A3-C1-C2 domains) is metal ion-dependent with residues in the A1 and A3 domains, which contain the interactive sites. Both Ca (II) and Mn (II) support this interaction, which is of interest since Ca (II) sites are typically formed by carboxylate moieties while Mn (II) can bind carboxylates as well as coordinated histidine residues. The divalent metal ion binding site (s) required for subunit association has (have) not been identified.

The liver is the major site of FVIII synthesis. The initial stage of secretion involves the translocation of the mature 2332 amino acids polypeptide into the lumen of the endoplasmic reticulum (ER) , where glycosylation occurs. Within the ER, FVIII appears to interact with a number of chaperone proteins, including calreticulin, calnexin and the IgG-binding protein. Due to the interaction with these chaperone proteins, a significant proportion of the FVIII molecules is retained within the ER, thereby limiting the transport of FVIII to the Golgi apparatus. The mechanism responsible for the transport from the ER to the Golgi apparatus is not elucidated yet. The activation of FVIII coincides with proteolysis of both the heavy and light chain. Cleavage within the heavy chain after arginine residue 740 generates a 90 kDa polypeptide, which is subsequently cleaved after arginine residue 372 to generate polypeptides of 50 and 43 kDa, whereas, the 80 kDa light chain is cleaved after arginine residue 1689 to generate a 73 kDa polypeptide.

FVIII becomes a potent cofactor of factor IXa once it is cleaved within the heavy and light chain. It is a cofactor for factor IXa, which, in the presence of Ca2+ and phospholipids, forms a complex that converts factor X to the activated form Xa. FVIII gene can also produce another alternative spliced variant, which encodes a putative small protein consisting primarily of the phospholipid binding domain of factor VIIIc.

RECOMBINANT AAV VECTOR

Adeno-associated virus (AAV) belong to the parvovirus, a single strand DNA (ssDNA) virus. The full-length genome of the AAV contains approximately 4.7 kilobases (kb) , comprising inverted terminal repeats (ITR) DNA sequences at both ends of the virus encompassing two open reading frames (ORF) called rep and cap.

The “AAV inverted terminal repeat (ITR) ” sequence is a sequence of about 145 nucleotides that exists at both ends of the natural single-stranded AAV genome. ITR is required for the efficient replication of the genome nucleic acid sequences of the symmetrical AAV particles, which can be used as a viral DNA synthesis origin of replication and are necessary structural components for the recombinant AAV vector.

The “rep” gene contains polynucleotide sequences encoding four rep proteins rep78, rep68, rep52, and rep40 required for the life cycle of AAV. The “cap” gene contains polynucleotide sequences encoding the AAV capsid proteins VP1, VP2, and VP3 proteins. The AAV capsid proteins VP1, VP2 and VP3 are capable to form a 24-subunit symmetrical AAV capsid through interaction between them.

AAV can effectively infect dividing and non-dividing human cells, and its genome can be integrated into a single chromosomal site in the host genome. Most importantly, although AAV already exists in humans, current research believes that AAV is not related to any disease. Based on its high safety, low immunogenicity, broad host range, ability to mediate stable long-term expression of exogenous genes in vivo, AAV has become the most promising vector system in gene therapy.

To date, 13 AAV serotypes has been identified according to the tissues or different cell types they infect. Further, according to the TABLE 1 below, different AAVs have been developed as advantageous vector systems for transfection of specific cell types. Among the many AAV serotypes, AAV2, AAV5, AAV8 and AAV9 are the most widely studied and used AAV vectors. They could transduce, including but not limited to, retinal epithelium, photoreceptor cells, skeletal muscle, central nervous system and/or liver cells; and have been used as carriers for many clinical tails in progress.

TABLE 1: AAV Serotypes and the Target Tissues Used in Gene Therapy

The term “recombinant AAV vector (rAAV vector) ” as used herein refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV) flanked by two AAV ITR sequences. When present in host cells expressing AAV rep and cap proteins, the rAAV vector can replicate and be packaged into AAV virus particles.

The term “recombinant AAV (rAAV) virus” or “rAAV viral particle” as used herein refers to rAAV vector encapsulated by at least one AAV capsid protein into AAV viral particles. Currently used host cells for rAAV viral particle production is derived from mammalian cell types, such as human embryonic kidney 293 (HEK293) cells and their derivatives such as HEK293T cells, human fibrosarcoma (HT-1080) cells, differentiated hepatocyte-derived carcinoma (Huh-7) cells, or PER. C6 cells, COS cells, murine myeloma (NS0) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, KB cells, and other mammalian cell lines. The rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell. However, the productivity of the virus of most of the above mammalian cell culture systems is insufficient in meeting the requirements of clinical trials and commercial scale production. To this end, an rAAV virus particle production system using insect cells such as Sf9 cells has recently been developed. However, to produce AAV in insect cells, some modifications must be made to obtain the correct stoichiometric ratio of the AAV capsid protein.

Baculovirus is a double-stranded circular DNA virus, belonging to Baculoviridae virus family, and has a genome size of 90 kb-230 kb. Baculoviruses are parasites exclusive to arthropods and known to infect more than 600 species of insects. Using Autographa Californica Multicapsid Nuclear Polyhedrosis Virus (AcMNPV) , Smith et al. successfully expressed human beta-interferon in the Sf9 cell line in 1983, developing the first baculovirus expression system (Mol. Cell Biol., 1983, 3: 2156-2165) . Since then, the baculovirus expression system has been continuously improved and developed, and it has become a very widely used eukaryotic expression system. In 2002, Urabe et al. showed that the baculovirus-infected Sf9 insect cell can support AAV replication, using three recombinant baculoviruses carrying AAV's rep gene, cap gene, and ITR core expression elements to co-infect the Sf9 cells and successfully prepared rAAV virus particles (Hum. Gene Ther. 2002; 13: 1935–1943) . On this basis, researchers have successively developed systems that are more suitable for large-scale preparation of rAAV virus particles.

At present, there are mainly two methods for large-scale preparation of rAAV virus particles using the baculovirus expression system: Two Bac system and One Bac system. The main process of the two baculovirus systems prepared rAAV viral particles is that the rep gene and the cap gene of the AAV is integrated into one baculovirus genome, and the ITR core element expressing the gene of interest is integrated into another baculovirus genome. The above two recombinant baculoviruses are then used to co-infect host cells to produce rAAV virus particles carrying the target gene. The main process of using One Bac system relies on packaging cell lines to prepare rAAV virus particles. A packaging cell line that can induce the expression of rep genes and cap genes is first established. This packaging cell line is integrated with the rep genes and cap gene expression elements. The rep gene and the cap gene are both placed under the control of the strong baculovirus late gene expression promoter polyhedrin (polh) . The hr2 enhancer sequence and the AAV rep protein binding sequence are further inserted upstream of the polh promoter. After being infected with a recombinant baculovirus containing AAV ITR and the target gene, the rep gene and cap gene in the packaging cell line are induced, and the rAAV virus particles containing the target gene insert are produced.

In some instances, the rAAV vector used to carry the gene of interest in the rAAV virus particle may also include one or more “expression control elements” . The term “expression control element” or “expression regulatory element” as used herein refers to a nucleic acid sequence that affects the expression of an operably linked polynucleotide, including polynucleotide sequences that promote the transcription and translation of heterologous polynucleotides. The expression control elements that can be used in the present disclosure include, but are not limited to, promoters, enhancers, intron splicing signals, poly A sequences, or inverted terminal repeats (ITR) .

A “promoter” is a DNA sequence located adjacent to a heterologous polynucleotide sequence encoding a target product, which is usually operably linked to an adjacent sequence, such as a heterologous polynucleotide. Compared to the amount expressed in the absence of a promoter, a promoter generally increases the amount of heterologous polynucleotide expression.

Examples of promoter include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron. ) , NSE (neuronal specific enolase) , synapsin or NeuN promoters, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP) ; a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE) , SFFV promoter, rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Other promoters can be of human origin or from other species, including from mice. Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, [beta] -actin, rat insulin promoter, the phosphoglycerate kinase promoter, the human alpha-1 antitrypsin (AAT) promoter, the transthyretin promoter (TTR) , the TBG promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EF1-alpha promoter, the CAG promoter and other constitutive promoters, hybrid promoters with multi-tissue specificity, promoters specific for neurons like synapsin and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, also find use herein. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif. ) . Some promoters, such as HLP, LP1, HCR-hAAT, ApoE-hAAT, and LSP, are described in more detail in the following references: HLP: Mcintosh J. et al, Blood 2013 Apr 25, 121 (17) : 3335-44; LP1: Nathwani et al, Blood. 2006 April 1, 107 (7) : 2653-2661; HCR-hAAT: Miao et al, Mol Ther. 2000; 1: 522-532; ApoE-hAAT: Okuyama et al, Human Gene Therapy, 7, 637-645 (1996) ; and LSP: Wang et al, Proc Natl Acad Sci U S A. 1999 March 30, 96 (7) : 3906-3910. Promoter is also described in WO 2011/005968. In some cases, the promoter is a liver specific promoter.

An “enhancer” is a sequence that enhances the activity of a promoter. Different from the promoter, an enhancer does not have the promoter activity, and may generally depend on its location relative to the promoter (i.e., upstream or downstream of the promoter) . Non-limiting examples of enhancer elements (or portions thereof) that can be used in the present disclosure include baculovirus enhancers and enhancer elements found in insect cells.

A “stuffer sequence” refers to a nucleotide sequence of a larger nucleic acid molecule (such as, but not limited, to a vector) , and is usually to create a desired gap or separation between two nucleic acid features (such as, but not limited, between a promoter and a coding sequence) or to extend the nucleic acid molecule a desired length. The stuffer sequence does not contain protein coding information and may have unknown or synthetic origin, not related to other nucleic acid sequences within the larger nucleic acid molecule, or any combination thereof.

As used herein, the term “intron” generally refers to a DNA molecule that may be isolated or identified from a gene and may be defined generally as a region spliced out during messenger RNA (mRNA) processing prior to translation. Alternately, an intron may be a synthetically produced or manipulated DNA element. An intron may contain enhancer elements that effect the transcription of operably linked genes. An intron may be used as a regulatory element for modulating expression of an operably linked transcribable DNA molecule. A construct may comprise an intron, and the intron may or may not be heterologous with respect to the transcribable DNA molecule.

In some embodiments, the enhancer/promoter that is operably connected to the coding sequence of FVIII is: apolipoprotein E locus hepatic control region (HCR) , human alpha-1-antitrypsin promoter (AAT) , transthyretin promoter (TTR) , or a combination thereof.

Virus Particle

The current disclosure provides a recombinant adeno-associated virus (rAAV) particle. The rAAV particle comprises a nucleic acid encoding a human FVIII protein without a B domain. In some embodiments, the nucleic acid comprises a SQ variant. In some other embodiments, the nucleic acid comprises a F309S mutation in the SQ variant. In some embodiments, the nucleic acid is a recombinant nucleic acid, which is any nucleic acid molecule containing joined nucleic acid molecules from different original sources and not naturally attached together. In some embodiments, the nucleic acid is synthetic nucleic acid, which may be synthesized in the laboratory. A recombinant nucleic acid as used herein can be DNA, or RNA. A recombinant nucleic acid can be prepared by using recombinant DNA technology by using enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. A recombinant DNA may be transcribed in vitro, to generate a messenger RNA (mRNA) , the recombinant mRNA may be isolated, purified and packaged into a rAAV particle.

Table 2 A list of amino acid sequences of hFVIII wild-type with B domain and hFVIII variant without B-domain (FVIII-SQ, also called FVIII-BDD) .

The polynucleotide disclosed herein comprises a nucleic acid encoding a human Factor VIII (FVIII) peptide. The FVIII peptide can lack a B domain. In some embodiments, the nucleic acid encoding the human FVIII peptide comprises a sequence with at least about 70%sequence identity to a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the human FVIII peptide comprises a sequence with at least about 75%sequence identity to a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the human FVIII peptide comprises a sequence with at least about 80%sequence identity to a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the human FVIII peptide comprises a sequence with at least about 85%sequence identity to a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the human FVIII peptide comprises a sequence with at least about 90%sequence identity to a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the human FVIII peptide comprises a sequence with at least about 95%sequence identity to a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the human FVIII peptide comprises a sequence with at least about 99%sequence identity to a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the human FVIII peptide comprises or consists of a sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the polynucleotide comprises a sequence with at least about 70%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 5-22 listed in Table 3. In some embodiments, the polynucleotide comprises a sequence with at least about 75%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 5-22. In some embodiments, the polynucleotide comprises a sequence with at least about 80%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 5-22. In some embodiments, the polynucleotide comprises a sequence with at least about 85%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 5-22. In some embodiments, the polynucleotide comprises a sequence with at least about 90%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 5-22. In some embodiments, the polynucleotide comprises a sequence with at least about 95%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 5-22. In some embodiments, the polynucleotide comprises a sequence with at least about 99%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 5-22. In some embodiments, the polynucleotide comprises or consists of a sequence selected from group consisting of SEQ ID NOs: 5-22.

Table 3. A list of nucleotide sequences of various constructs for encoding FVIII protein variants.

In some embodiments, the polynucleotide comprises a sequence with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or any percentage between the aforementioned percentages sequence identity to a sequence selected from group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 22. In some embodiments, the polynucleotide comprises a sequence with at least about 90%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 22. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22.

In some embodiments, the polynucleotide comprises a sequence with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or any percentage between the aforementioned percentages sequence identity to SEQ ID NOs: 6, 10, 18, or 20. In some embodiments, the polynucleotide comprises a sequence with at least about 90%sequence identify to SEQ ID NOs: 6, 10, 18, or 20. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NOs: 6, 10, 18, or 20. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 6. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 10. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 20.

In some embodiments, the polynucleotide comprises a sequence with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or any percentage between the aforementioned percentages sequence identity to a sequence selected from group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, and 22. In some embodiments, the polynucleotide comprises a sequence with at least about 90%sequence identity to a sequence selected from group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, and 22. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NOs: 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

In some embodiments, the polynucleotide comprises a sequence with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or any percentage between the aforementioned percentages sequence identity to SEQ ID NOs: 17, 18, 19, 20, 21, or 22. In some embodiments, the polynucleotide comprises a sequence with at least about 90%sequence identify to SEQ ID NOs: 17, 18, 19, 20, 21, or 22. In some embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NOs: 17, 18, 19, 20, 21, or 22.

In some embodiments, the expression/secretion level of the FVIII peptide encoded by the polynucleotide disclosed herein is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%compared to an expression/secretion level of a wild type FVIII peptide without a B domain encoded by a wild-type nucleic acid. The increased expression/secretion of FVIII peptide may reduce endoplasmic reticulum (ER) stress response. FVIII or FVIII-SQ is a protein known to be difficult to fold and secrete, this could potentially induce endoplasmic reticulum (ER) and activate unfolded protein response (UPR) . This process may damage the cells, leading to decreased expression of FVIII or FVIII-SQ. The endoplasmic reticulum (ER) is a multifunctional organelle essential for the synthesis, folding, and processing of secretory and transmembrane proteins. In order for proteins to fold properly a balance between the ER protein load and the folding capacity to process this load must be established. However, physiological and pathological stimuli can disrupt this ER homeostasis resulting to an accumulation of misfolded and unfolded proteins, a condition known as ER stress. ER stress activates a complex signaling network referred as the UPR to reduce ER stress and restore homeostasis. However, if the UPR fails to reestablish the ER to normality, ER stress causes cell dysfunction and death. The adaptive response to ER stress is the UPR. The UPR is initiated by three ER transmembrane proteins: Inositol Requiring 1 (IRE1) , PKR-like ER kinase (PERK) , and Activating Transcription Factor 6 (ATF6) . During unstressed conditions, the ER chaperone, immunoglobin binding protein (BiP) binds to the luminal domains of these master regulators keeping them inactive. Upon ER stress, BiP dissociates from these sensors resulting to their activation. Thus, activation and expression levels of the various regulators, such as IRE1, PERK, ATF6, etc. may be used to measure the ER response and the UPR. Moreover, the ER lumen size can be measured to detect ER stress. In addition, an increase in ER protein load could overwhelm oxidative folding enzymes, preventing proper disulfide formation and therefore inducing ER stress. Without being limited to any specific theory, the FVIII and FVIII-SQ with a F309S mutation can significantly increase its protein secretion out of cells and reduce ER stress in the cells, which may increase durability of FVIII gene therapy efficacy.

In some embodiments, the hFVIII peptide encoded by nucleic acid disclosed herein reduces an ER stress response by at least 5%, 10%, 15%, 20%, 25%, or 30%, 35%, 40%, 45%, or 50%compared to that of wild-type hFVIII peptide without B-domain. In some embodiments, the increased secretion of hFVIII peptide encoded by nucleic acid disclosed herein reduces a UPR by at least 5%, 10%, 15%, 20%, 25%, or 30%, 35%, 40%, 45%, or 50%compared to that of wild-type hFVIII peptide without B-domain.

In some embodiments, disclosed herein is a polynucleotide encoding a AAV capsid protein comprising a first promoter, a first ORF, a second promoter, and a second ORF. The first promoter, the first ORF, the second promoter, and the second ORF may be in any suitable order for expressing the first ORF and the second ORF, respectively. In some embodiments, the polynucleotide comprising the first promoter, the first ORF, the second promoter, and the second ORF, from 3’ to 5’, is in an order as follows: the first promoter, the first ORF comprising the second promoter, and the second ORF, wherein the second ORF comprises at least one translation start codon and overlaps with the 3’ portion of the first ORF. In some embodiments, the second ORF comprises at least one initial translation codon and not with the first ORF of the 3’ overlapping portion. In some embodiments, the first ORF, the first promoter, the second ORF, and the second promoter from 3' to 5 ' sequence is as follows: first promoter, second promoter comprising a first ORF, a second ORF, wherein the second ORF comprises at least one initial translation codon and the first ORF of the 3’ overlapping portion.

In some embodiments, the polynucleotide is codon optimized. In some embodiments, the nucleic acid is codon optimized. In some embodiments, the nucleic acid encoding the hFVIII comprises a reduced number of CpG dinucleotides as compared to the wild type hFVIII coding sequence. In some embodiments, the nucleic acid encoding the hFVIII comprises less than 100, less than 80, lessen than 60, less than 40, less than 20, less than 15, less than 10, or less than 5 CpG dinucleotides. In some embodiments, the nucleic acid encoding the hFVIII comprises not more than 50%of CpG as compared to the wild type hFVIII coding sequence. In some embodiments, the nucleic acid encoding the hFVIII comprises from 50 to 100 CpG dinucleotides. In some embodiments, the polynucleotide comprises not more than 200 or not more than 100 CpG dinucleotides. In some embodiments, the polynucleotide comprises from 200 to 500 CpG dinucleotides. In some embodiments, the polynucleotide disclosed herein has a reduced immunogenicity compared to a corresponding polynucleotide comprising a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain. In some embodiments, the nucleic acid disclosed herein has a reduced immunogenicity compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain.

In some embodiments, the recombinant rAAV protein comprises an AAV cap protein. In some embodiments, the AAV cap protein can be any structural protein known in the art that can form a functional AAV capsid (i.e., packaging DNA and infecting target cells) . In some embodiments, the cap protein includes VP1, VP2, and VP3. In some embodiments, the cap protein does not need to comprise all of VP1, VP2, and VP3, as long as it can produce a functional AAV capsid. In some embodiments, the cap protein includes VP1 and VP2. In some embodiments, the cap protein comprises VP1 and VP3. In some embodiments, the cap protein includes VP2 and VP3. In some embodiments, the cap protein comprises VP1. In some embodiments, the cap protein includes VP2. In some embodiments, the cap protein includes VP3.

VP1, VP2, or VP3 may be derived from any AAV serotype. In some embodiments, the VP1 may be derived from AAV serotype 1 (AAV1) , AAV serotype 2 (AAV2) , AAV2 variants, AAV serotype 3 (AAV3, including serotypes 3A and 3B) , AAV serotype 4 (AAV4) , the AAV serotype 5 (AAV5) , the AAV serotype 6 (AAV6) , the AAV serotype 7 (AAV7) , the AAV serotype 8 (AAV8) , the AAV serotype 9 (AAV9) , the AAV serotype 10 (AAV10) , AAV serotype 11 (AAV11) , AAV serotype 12 (AAV12) , AAV serotype 13 (AAV13) , AAV-Rh10, AAV-Rh74, AAV-2i8 or any other known AAVs. In some embodiments, the VP1 and the wildtype VP1 derived from AAV1, AAV2, AAV3, (including AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV2i8 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the VP1 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype VP1 derived from AAV1, AAV2, AAV3 (including AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, of AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74 or AAV-2i8.

In some embodiments, the VP2 may be derived from AAV serotype 1 (AAV1) , AAV serotype 2 (AAV2) , AAV2 variants, AAV serotype 3 (AAV3, including serotypes 3A and 3B) , AAV serotype 4 (AAV4) , the AAV serotype 5 (AAV5) , the AAV serotype 6 (AAV6) , the AAV serotype 7 (AAV7) , the AAV serotype 8 (AAV8) , the AAV serotype 9 (AAV9) , the AAV serotype 10 (AAV10) , AAV serotype 11 (AAV11) , AAV serotype 12 (AAV12) , AAV serotype 13 (AAV13) , AAV-Rh10, AAV-Rh74, AAV-2i8 or any other known AAVs. In some embodiments, the VP2 and the wildtype VP2 derived from AAV1, AAV2, AAV3 (including AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV2i8 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the VP2 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype VP2 derived from AAV1, AAV2, AAV3 (including AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, of AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74 or AAV-2i8.

The VP3 may be derived from AAV serotype 1 (AAV1) , AAV serotype 2 (AAV2) , AAV2 variants, AAV serotype 3 (AAV3, including serotypes 3A and 3B) , AAV serotype 4 (AAV4) , the AAV serotype 5 (AAV5) , the AAV serotype 6 (AAV6) , the AAV serotype 7 (AAV7) , the AAV serotype 8 (AAV8) , the AAV serotype 9 (AAV9) , the AAV serotype 10 (AAV10) , AAV serotype 11 (AAV11) , AAV serotype 12 (AAV12) , AAV serotype 13 (AAV13) , AAV-Rh10, AAV-Rh74, AAV-2i8 or any other known AAVs. In some embodiments, the VP3 and the wildtype VP3 derived from AAV1, AAV2, AAV2 variants, AAV3, (including AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV2i8 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the VP3 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype VP3 derived from AAV1, AAV2, AAV2 variants, AAV3 (including AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, of AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74 or AAV-2i8.

In some embodiments, the cap protein comprises VP1, VP2, VP3, or any combinations thereof derived from AAV of the same serotype; for example, the cap protein may comprise VP1, VP2, VP3, or any combinations thereof derived from AAV2, AAV2 variants, AAV5, AAV6, AAV8, AAV9, AAV9 variants, AAV-DJ, LK03, NP59, and KP1. In some embodiments, the cap comprises VP1, VP2, VP3, or any combinations thereof derived from different serotypes of AAV; for example, the cap protein may comprise any one or more of VP1, VP2, VP3, or any combination thereof of AAV1, AAV2, AAV2 variants, AAV3 (including AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAV-DJ, LK03, NP59, or KP1.

In some embodiments, the cap protein may be cloned into pUC57, pFastBac1, modified pUC57, or modified pFastBac1. In some embodiments, the cap protein may be cloned into pUC57. In some embodiments, the cap protein may be cloned into pFastBac1. In some embodiments, the cap protein may be cloned into modified pUC57. In some embodiments, the cap protein may be cloned into modified pFastBac1.

In some embodiments, the polynucleotide sequence encoding the cap protein is operably linked to a first promoter. The first promoter may be any suitable promoter known in the art that can drive the expression of the cap protein. In some embodiments, the first promoter may be a tissue-specific promoter, a constitutive promoter, or a regulatable promoter. In some embodiments, the first promoter can be selected from different sources, for example, the first promoter can be a viral promoter, a plant promoter, or a mammalian promoter.

In some embodiments, the 3’ end of a DNA sequence, such as, for example, the cap protein sequence or the FVIII sequence, further comprises a polyadenylation sequence or “poly A sequence. ” In some embodiments, the 3’ end of the FVIII further comprises a polyadenylation sequence or “poly A sequence. ” In some embodiments, the polyadenylation sequences or “poly A sequences” may range from about l to about 500 base pairs (bp) . In some embodiments, the polyadenylation sequence or “poly A sequence” may be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 200, or 500 nucleotides.

In some embodiments, the first ORF operably linked to the first promoter. The first promoter may be any suitable promoter known in the art that can drive the expression of the cap. In some embodiments, the first promoter may be a tissue-specific promoter, a constitutive promoter, or a regulatable promoter. In some embodiments, the first promoter can be selected from different sources, for example, the first promoter can be a viral promoter, a plant promoter, and a mammalian promoter.

Examples of such promoters include but are not limited to, the human cytomegalovirus (CMV) immediate-early increase hadron /promoter, the SV40 early enhancer /promoter, the JC polyomavirus promoter, the myelin basic protein (MBP) or glial fibrillar acidic protein fibers white (GFAP) promoter, herpes simplex virus (HSV-1) latency-associated promoter (LAP) , Rous sarcoma virus (RSV) long terminal repeat (LTR ) promoter, neuron-specific promoters (of NSE) , platelet-derived growth factor (PDGF) promoter, HSYN , melanin-concentrating hormone (MCH) promoter, CBA, the protein matrix metal promoter (MPP) , chicken beta-Actin promoter, CAG, MNDU3, PGK, EF1a, and liver-specific promoters.

In some embodiments, the promoter is a promoter suitable for expression in insect cells. In some embodiments, the suitable for expression in an insect cell promoters include, but are not limited to polh promoter, p10 start promoter, alkaline promoter, an inducible promoter, E1 promoter or a Delta E1 promoter. In some embodiments, the promoter is the polh promoter. In some embodiments, the promoter is the p10 promoter.

In some embodiments, the first ORF of the 3' end further comprises a polyadenylation sequence or "poly A sequence. " The length of the polyadenylation sequence or "poly A sequence" may range from about 1 to 500 bp. The polyadenylation sequence or "poly A sequence length" may be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 200 or 500 nucleotides.

In some embodiments, the said second ORF encodes the AAV rep protein, wherein the rep protein can be any rAAV vector required for replication and packaging of rAAV viral particles. In some embodiments, the rep protein includes rep 78, rep 68, rep 52 and rep 40. In some embodiments, the rep protein need not include all of rep 78, rep 68, rep 52, and rep 40, as long as it can allow the rAAV vector to replicate and package into rAAV virus particles. In some embodiments, the rep protein includes any three of rep 78, rep 68, rep 52 and rep 40. In some embodiments, the rep proteins include any two of rep 78, rep 68, rep 52 and rep 40. In some embodiments, the rep protein includes any one of rep 78, rep 68, rep 52 and rep 40. In some embodiments, the rep protein includes rep 78 and rep 52. In some embodiments, the rep protein includes rep 78 and rep 40. In some embodiments, the rep protein includes rep 68 and rep 52. In some embodiment, the rep protein includes rep 68 and rep 40.

The rep 78, rep 68, rep 52 and the rep 40 may be derived from any AAV serotype. In some embodiments, the rep 78 may be derived from AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, the AAV-Rh10, AAV-Rh74, AAV-2i8 and any other known AAV. In some embodiments, the rep 78 may be derived from AAV2 or the AAV 5. In some embodiments, the rep 78 and derived from AAV1, AAV2, AAV3, (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74 or AAV-2i8 wild type REP 78 at least 75%, 80%, 85%, 90%, 95%or more identity. In some embodiments, the rep 78 and from AAV2 or the AAV5 wild type rep 78 at least 75%, 80%, 85%, 90%, 95%or more identity. In some embodiments, the rep 78 compared from AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, the AAV-Rh10, the AAV -Rh74 or AAV-2i8 wildtype rep 78 has a one or more substituted amino acids, deletions and /or additions. In some embodiments, the rep 78 compared from AAV2 or the AAV5 wild type rep 78 has a one or more amino acid substitutions, deletions and /or additions.

In some embodiments, the rep 68 may be derived from AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8 and any other known AAV. In some embodiments, the rep 68 can be derived from AAV2 or AAV5. In some embodiments, the rep 68 derived from AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, the AAV-Rh10, the AAV-Rh74 or AAV-2i8 wild type rep 68 has at least 75%, 80%, 85%, 90%, 95%or more sequence identity thereof. In some embodiments, the rep 68 derived from AAV2 or the AAV5 has at least 75%, 80%, 85%, 90%, 95%or more sequence identity thereof. In some embodiments, the derived rep 68 has one or more amino acid substitutions, deletions and /or additions as compared to the wild-type AAV1, AAV2, AAV3, (including the AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74 or AAV-2i8. In some embodiments, the rep 68 has a one or more amino acid substitutions, deletions and /or additions as compared to a wild type rep 68 from AAV2 or the AAV5.

In some embodiments, the rep 52 may be derived from AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8 and any other known AAV. In some embodiments, the rep 52 may be derived from AAV2 or the AAV5. In some embodiments, the said rep 52 derived from the AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, A AV13, the AAV-Rh10, the AAV-Rh74 or AAV-2i8 wild type rep 52 has at least 75%, 80%, 85%, 90%, 95%or more sequence identity thereof. In some embodiments, the rep 52 derived from AAV2 or the AAV5 wild type rep 52 has at least 75%, 80%, 85%, 90%, 95%or more sequence identity thereof. In some embodiments, the rep 52, as compared to the AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74 or AAV-2i8 wild type rep 52, has one or more amino acid substitutions, deletions and /or additions. In some embodiments, the rep 52, as compared to AAV2 or AAV5 wild type rep 52, has one or more amino acid substitutions, deletions and/or additions.

In some embodiments, the rep 40 is derived from AAV1, of AAV2, AAV3 (including the AAV 3A and 3B ) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8 and any other known AAV. In some embodiments, the rep 40 is derived from AAV2 or AAV5. In some embodiments, the rep 40 derived from AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74 or AAV-2i8 wild type rep 40 has at least 75%, 80%, 85%, 90%, 95%or more sequence identity thereof. In some embodiments, the rep 40 derived from AAV2 or AAV5 wild type rep 40 has at least 75%, 80%, 85%, 90%, 95%or more sequence identity thereof. In a some embodiments, the rep 40, as compared to AAV1, AAV2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74 or AAV-2i8 wild type rep 40, has one or more amino acid substitutions, deletions and/or additions. In some embodiments, the rep 40, as compared to AAV2 or AAV5 wild type rep 40, has a one or more substituted amino acids, deletions and/or additions.

In some embodiments, the rep includes units derived from the same serotype AAV including rep 78, rep 68, rep 52 and/or rep 40. In some embodiments, the rep comprises units derived from AAV2 including rep 78, rep 68, rep 52 and/or rep 40. In some embodiments, the rep comprises units derived from AAV2 including rep 78 and/or rep 52. In some embodiments, the rep comprises units derived from AAV5 including rep 78 , rep 68 , rep 52 and/or rep 40. In some embodiments, the rep comprises units derived from AAV5 including rep 78 and/or rep 52. In some embodiments, the rep comprises units derived from different serotypes of AAVs including rep 78 , rep 68 , rep 52 and/or rep 40. For example, the rep may comprise units derived from any rep 78 , rep 68 , rep 52 and /or rep 40 of AAV1, AAV2, AAV3 (including AAV3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AV12, AAV13, AAV-Rh10, AAV-Rh74, and/or AAV-2i8.

In some embodiments, the second ORF encoding the rep protein is operably linked to a second promoter. The second promoter may be any suitable promoter known in the art that can drive the expression of the cap. In some embodiments, the second promoter is a tissue-specific promoter, a constitutive promoter, or a regulatable promoter . In some embodiments, the second promoter is selected from different sources, for example, the second promoter may be a viral promoter, plant promoter and a mammalian promoter.

In some embodiments, the rAAV particle comprises a second promoter. The examples of the second promoter described below include, but are not limited to, human cytomegalovirus (CMV) immediate-early enhancer/promoter, SV40 early enhancer/promoter, JC polyomavirus promoter, myelin basic Protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency-related promoter (LAP) , Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuron-specific promoters (NSE) , platelet-derived growth factor (PDGF) promoter, HSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloproteinase white matter promoter (MPP) , chicken β -actin promoter, CAG, MNDU3, PGK and EF1a promoter.

In some embodiments, the second promoter is a promoter suitable for expression in insect cells. In some embodiments, the suitable promoters for expression in an insect cell include, but are not limited to, polh promoter, p10 promoter, basic promoters, inducible promoters, E1 promoters, or Delta E1 promoter. In some embodiments, the second promoter is polh promoter. In some embodiments, the second promoter is the p10 promoter.

In some embodiments, the second ORF's 3' end further comprises a polyadenylation sequence or "poly A sequence. " In some embodiments, the polyadenylation of a sequence or "poly A sequence" may range from about l-500 BP. In some embodiments, the polyadenylation acidified sequence or "poly A sequence length" may be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 200 or 500 nucleotides.

In some embodiments, the cap and the rep are derived from the same AAV serotype. For example, the cap and rep may be derived from the same AAV1, AAV 2, AAV3 (including the AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8 or any other known AAV.

In some embodiments, the cap and the rep are derived from different AAV serotypes, e.g., the cap and the rep may be derived respectively from AAV1, AAV2, AAV3 (including AAV 3A and 3B) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh 10, AAV-Rh74, AAV-2i8, or any other known AAV. For example, in some embodiments, the cap is derived from AAV2 and the rep is derived from AAV5.

In some embodiments, the second ORF comprises at least one initial translation codon and the 3 overlapping 'portion of the first ORF. In some embodiments, the first ORF comprises an intron sequence containing the second promoter. In some embodiments, the first ORF and the second ORF are linked by a sequence encoding a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the splitable solution linker as comprising 2A sequence of the peptide. In some embodiments, the 2A peptide is selected from 2A peptides derived from Aphthoravirus or Cardiovirus , such as foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV) , Thoseaasigna virus (TaV) or the 2A peptide of porcine teschovirus 1 (PTV-1) .

In some embodiments, the second polynucleotide comprises with the CMV, CAG, MNDU3, PGK, EF1a promoter, or liver unit-specific promoter operably linked transgene. In some embodiments, the liver-specific promoter comprises one or more of the following: a TTR promoter, AAT promoter, albumin (ALB) promoter, apolipoprotein protein (APO) promoter, apolipoprotein A (apolipoprotein A, APOA1) promoter, apolipoprotein a C3 (apolipoprotein a C3, APOC3) promoter, complement factor B (complement factor B, CFB) promoter, keto hexose kinase (Ketohexokinase, KHK) promoter, hemagglutinin (Hemopexin , HPX) promoter, nicotinamide N-methyltransferase (ICOT inamide N -methyltransferas E, NNMT) promoter or minimal promoter, (liver) carboxylic acid esterase 1 (Carboxylesterase. 1, CES1) promoter, protein C (protein C, the PROC) promoter, mannan-binding lectin serine protease 2 (mannan-binding lectin serine protease 2, MASP-2) promoter, hepcidin antimicrobial Peptide (epcidin antimicrobial peptide, HAMP) promoter, or modified variants thereof.

In some embodiments, the liver-specific promoter further comprises other regulatory sequences. In some embodiments, the regulatory sequence comprises an enhancer sequence. In some embodiments, the enhancer sequence comprises a liver-specific control region in liver (liver-specific hepatic control region, the HCR) enhancer.

In some of the embodiments, the liver unit-specific promoter selected from the group of HCR-AAT, TTR, AAT-TTR, and its modified variants.

For producing the rAAV particles, the present disclosure provides a recombinant adeno-associated virus (rAAV) particle prepared by introducing or transfecting the composition of the present disclosure into a host cell. In some embodiments, the host cell is an insect cell, a human cell or an animal cell. In some embodiments, the insect cell is Drosophila S2 cells or Sf9 cells. In some embodiments, the animal cell is fibroblasts, Chinese hamster ovary (CHO) cells, COS cells, murine myeloma (NS0) cells, HeLa cells, Baby Hamster Kidney (BHK) cells. In some embodiments, the human cell is human embryonic kidney 293 (HEK293) cells, human fibrosarcoma (HT-1080) cells, differentiated hepatocyte-derived carcinoma (Huh-7) cells, or PER. C6 cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is HEK293 cell or its derivatives such as 293T cells. In some embodiments, the preparation includes, but is not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection. In some embodiments, the composition is transfected into the 293T cells with a helper plasmid. In some embodiments, the 293T cells are used to produce the rAAV virus particles.

In some embodiments, the composition of the present disclosure may be delivered into the insect cell by any method known in the art. In some embodiments, the method includes, but is not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and/or infection. In some embodiments, the composition is infected into the insect cell. In some embodiments, the composition is stably transfected into the insect cell.

In some embodiments, the method for preparing recombinant AAV virus particles may comprise generating bacmid DNA and/or baculovirus. In some embodiments, the method for preparing recombinant AAV virus particles may comprise generating FVIII expression sequence bacmid DNA. In some embodiments, the method for preparing recombinant AAV virus particles may comprise generating rAAV rep-cap expression sequence bacmid DNA. In some embodiments, the method for preparing recombinant AAV virus particles may comprise transfecting a host cell with the bacmid DNA to produce baculoviruses. In some embodiments, the method for preparing recombinant AAV virus particles may comprise transfecting a host cell with the FVIII expression sequence bacmid DNA to produce baculoviruses. In some embodiments, the method for preparing recombinant AAV virus particles may comprise transfecting a host cell with the rAAV rep-cap expression sequence bacmid DNA to produce baculoviruses. In some embodiments, the method for preparing recombinant AAV virus particles may further comprise mixing the two baculoviruses to infect a host cell (such as the Sf9 cell) to obtain packaged rAAV/FVIII-optimized virus particles of the present disclosure.

In some embodiments, the method for preparing recombinant AAV virus particles comprises (1) generating FVIII expression sequence bacmid DNA, (2) generating rAAV rep-cap expression sequence bacmid DNA, (3) transfecting a host cell with the bacmid DNA to produce baculoviruses, (4) transfecting a host cell with the FVIII expression sequence bacmid DNA to produce baculoviruses, (5) transfecting a host cell with the rAAV rep-cap expression sequence bacmid DNA to produce baculoviruses, and (6) mixing the two baculoviruses to infect a host cell (such as the Sf9 cell) to obtain packaged rAAV/FVIII-optimized virus particles of the present disclosure.

In some cases, the rAAV virus particles can be isolated and purified from the insect cells according to conventional methods known to those skilled in the art. For example, the rAAV can be purified using centrifugation, HPLC, hydrophobic interaction chromatography (HIC) , anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, other purification techniques, or any combinations thereof.

The rAAV viral particles comprises a polynucleotide comprising a nucleic acid encoding a human Factor VIII peptide lacking a B domain, wherein the Factor VIII peptides comprises a F309S mutation. The rAAV particle can comprise an AAV capsid protein and the AAV capsid protein can be AAV2, AAV3B, AAV5, AAV6, AAV8, AAV3BAAV9, AAVHSC, AAVDJ, LK03, NP59, or KP1. The rAAV particle can be a single-stranded AAV (ssAAV) . In some instances, the rAAV viral particles comprises a polynucleotide comprising any of the nucleic acid encoding the human FVIII as disclosed herein. In some instances, the rAAV viral particles comprises any polynucleotide disclosed herein. In some instances, the rAAV viral particles comprises a polynucleotide with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5%sequence identity of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22. In some instances, the rAAV viral particles comprises a polynucleotide with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5%sequence identity of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 21. In some instances, the rAAV viral particles comprises a polynucleotide with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5%sequence identity of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 21. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 21. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 21. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 5. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 6. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 9. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 10. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 12. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 15. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 16. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 18. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 20. In some instances, the rAAV viral particles comprises a polynucleotide with at least 90%sequence identity of SEQ ID NO: 21. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 5. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 6. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 9. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 10. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 12. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 15. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 16. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 18. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 20. In some instances, the rAAV viral particles comprises a polynucleotide with at least 95%sequence identity of SEQ ID NO: 21. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 5. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 6. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 9. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 10. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 12. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 15. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 16. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 18. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 20. In some instances, the rAAV viral particles comprises a polynucleotide of SEQ ID NO: 21.

In some instances, the rAAV comprises a capsid protein of a liver targeting AAV. In some instances, the liver targeting AAV is KP1, AAV5, AAV7, AAV8, or AAV9. In some instances, the liver targeting AAV is AAV5. In some instances, the liver targeting AAV is AAV8.

In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 10, 18, or 20.

In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide selected from SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide selected from SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide selected from 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of KP1 and a polynucleotide selected from 6, 10, 18, or 20. In some instances, the KP1 is a KP1 variant.

In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 10, 18, or 20. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 6. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 10. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 18. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 20.

In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide selected from SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide selected from SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide selected from SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide selected from SEQ ID NOs: 6, 10, 18, or 20. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide of SEQ ID NO: 6. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide of SEQ ID NO: 10. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide of SEQ ID NO: 18. In some instances, the rAAV comprises a capsid protein of AAV5 and a polynucleotide of SEQ ID NO: 20. In some instances, the AAV5 is an AAV5 variant.

In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 10, 18, or 20. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 6. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 10. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 18. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 20.

In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide selected from SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide selected from SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide selected from SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide selected from SEQ ID NOs: 6, 10, 18, or 20. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide of SEQ ID NO: 6. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide of SEQ ID NO: 10. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide of SEQ ID NO: 18. In some instances, the rAAV comprises a capsid protein of AAV8 and a polynucleotide of SEQ ID NO: 20. In some instances, the AAV8 is an AAV8 variant.

In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 10, 18, or 20. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 6. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 10. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 18. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 20.

In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide selected from SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide selected from SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide selected from SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide selected from SEQ ID NOs: 6, 10, 18, or 20. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide of SEQ ID NO: 6. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide of SEQ ID NO: 10. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide of SEQ ID NO: 18. In some instances, the rAAV comprises a capsid protein of AAV7 and a polynucleotide of SEQ ID NO: 20. In some instances, the AAV7 is an AAV7 variant.

In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 10, 18, or 20. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 6. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 10. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 18. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide having at least 90%, 95%, or 99%of SEQ ID NO: 20.

In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide selected from SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide selected from SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide selected from SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide selected from SEQ ID NOs: 6, 10, 18, or 20. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide of SEQ ID NO: 6. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide of SEQ ID NO: 10. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide of SEQ ID NO: 18. In some instances, the rAAV comprises a capsid protein of AAV9 and a polynucleotide of SEQ ID NO: 20. In some instances, the AAV9 is an AAV9 variant.

In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide having at least 90%, 95%, or 99%of one selected from any of SEQ ID NOs: 18, 20, or 22.

In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide selected from SEQ ID NOs: 5-22. In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide selected from SEQ ID NOs: 5, 6, 9, 10, 12, 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide selected from SEQ ID NOs: 15, 16, 18, 20, or 21. In some instances, the rAAV comprises a capsid protein of AAV2 and a polynucleotide selected from SEQ ID NOs: 6, 10, 18, or 20.

The rAAV virus particles disclosed herein can be used to express the FVIII or the variant disclosed herein. In some cases, the FVIII or the variant is expressed in vivo. The expression of the FVIII from the rAAV virus can be increased compared to an expression level of a wild type FVIII peptide without a B domain encoded by a wild-type nucleic acid from a rAAV. In some embodiments, the expression level of the FVIII peptide expressed from the rAAV disclosed herein is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%compared to an expression level of a wild type FVIII peptide without a B domain encoded by a wild-type nucleic acid from a rAAV. The increased expression/secretion of FVIII peptide may reduce endoplasmic reticulum (ER) stress response. In some embodiments, the hFVIII peptide encoded by nucleic acid from a rAAV disclosed herein reduces an ER stress response by at least 5%, 10%, 15%, 20%, 25%, or 30%, 35%, 40%, 45%, or 50%compared to that of wild-type hFVIII peptide without B-domain from a rAAV. In some embodiments, the increased secretion of hFVIII peptide encoded by nucleic acid from a rAAV disclosed herein reduces a UPR by at least 5%, 10%, 15%, 20%, 25%, or 30%, 35%, 40%, 45%, or 50%compared to that of wild-type hFVIII peptide without B-domain from a rAAV.

The rAAV disclosed herein can express hFVIII peptide at a concentration from about 0.5 IU/mL to about 3.5 IU/mL after transducing Huh-7 cells using the KP1 capsid. The concentration is measured by a FVIII ELISA assay. In some instances, the rAAV expresses the hFVIII peptide at a concentration of about 1 IU/mL. In some instances, the rAAV expresses the hFVIII peptide at a concentration of about 2 IU/mL. In some instances, the rAAV expresses the hFVIII peptide at a concentration from about 1 IU/mL to about 2 IU/mL. In some instances, the rAAV expresses the hFVIII peptide at a concentration from about 0.5 IU/mL to about 1 IU/mL. In some instances, the rAAV expresses the hFVIII peptide at a concentration of about 3 IU/mL. In some instances, the rAAV expresses the hFVIII peptide at a concentration from about 2 IU/mL to about 3 IU/mL.

The rAAV disclosed herein can express hFVIII peptide at a plasma concentration from about 50%to about 300%post-dosing in the FVIII-KO mice using the KP1 capsid. The plasma concentration is calculated as based on FVIII concentration in ELISA assay standard. In some instances, the rAAV expresses the hFVIII peptide at a plasma concentration from about 100%to about 200%. In some instances, the rAAV expresses the hFVIII at a plasma concentration from about 120%to about 180%. In some instances, the rAAV expressed the hFVIII at a plasma concentration of about 150%. The rAAV can express the hFVIII at a plasma concentration from about 50%to about 300%about two weeks after initial dosing. In some instances, the rAAV expresses the hFVIII at a plasma concentration from about 100%to about 200%about two weeks after initial dosing. In some instances, the rAAV expresses the hFVIII at a plasma concentration from about 120%to about 180%about two weeks after initial dosing. In some instances, the rAAV expresses the hFVIII at a plasma concentration from about 150%about two weeks after initial dosing.

The rAAV can maintain an expression of the hFVIII peptide at a plasma concentration from about 20%to about 250%post-dosing in the FVIII-KO mice using the KP1 capsid. The rAAV can maintain the expression of the hFVIII at a plasma concentration from about 50%to about 200%. The rAAV can maintain the expression of the hFVIII at a plasma concentration from about 100%to about 150%. The rAAV can maintain the expression of the hFVIII at a plasma concentration of about 120%. The rAAV can maintain the expression of hFVIII at a plasma concentration from about 20%to about 250%about one month after initial dosing. The rAAV can maintain the expression of the hFVIII at a plasma concentration from about 50%to about 200%about one month after initial dosing. The rAAV can maintain the expression of the hFVIII at a plasma concentration from about 100%to about 150%about one month after initial dosing. The rAAV can maintain the expression of the hFVIII at a plasma concentration of about 120%about one month after initial dosing.

The hFVIII expressed from the rAAV disclosed herein can have an activity from about 0.5 IU/mL to about 3 IU/mL. In some instances, the activity is from about 1 IU/mL to about 2 IU/mL. In some instances, the activity is about 1 IU/mL. In some instances, the activity is about 2 IU/mL. In some instances, the activity is about 2.5 IU/mL. In some instances, the activity is from about 1 IU/mL to about 2.5 IU/mL. In some instances, the activity is from about 1.5 IU/mL to about 2.5 IU/mL. In some instances, the activity is from about 1.5 IU/mL to about 2 IU/mL.

The hFVIII expressed from the rAAV disclosed herein can have a specific activity (activity/protein) from about 0.5 to about 2. In some instances, the specific activity is from about 0.5 to about 1.5. In some instances, the specific activity is from about 1 to about 2. In some instances, the specific activity is about 1. In some instances, the specific activity is about 1.5. In some instances, the specific activity is from about 1 to about 1.5.

The hFVIII expressed from the rAAV disclosed herein can have a biological activity in plasma from about 25%to about 200%post-dosing in the FVIII-KO mice using the KP1 capsid. The biological activity in plasma is calculated based on FVIII activity in the control human plasma standard. In some instances, the expressed hFVIII can have a biological activity in plasma from about 25%to about 150%post-dosing. In some instances, the expressed hFVIII can have a biological activity in plasma from about 50%to about 120%post-dosing. In some instances, the expressed hFVIII can have a biological activity in plasma from about 80%to about 100%post-dosing. In some instances, the expressed hFVIII can have a biological activity in plasma of about 100%post-dosing. The expressed hFVIII can have a biological activity in plasma from about 25%to about 200%about two weeks after initial dosing. In some instances, the expressed hFVIII has a biological activity in plasma from about 25%to about 150%about two weeks after initial dosing. In some instances, the expressed hFVIII has a biological activity in plasma from about 50%to about 120%about two weeks after initial dosing. In some instances, the expressed hFVIII has a biological activity in plasma from about 80%to about 100%about two weeks after initial dosing. In some instances, the expressed hFVIII has a biological activity in plasma about 100%about two weeks after initial dosing.

The hFVIII expressed from the rAAV disclosed herein can maintain a biological activity in plasma from about 25%to about 150%post-dosing in the FVIII-KO mice using the KP1 capsid. In some instances, the expressed hFVIII maintains a biological activity in plasma from about 50%to about 150%post-dosing. In some instances, the expressed hFVIII maintains a biological activity in plasma from about 50%to about 100%post-dosing. In some instances, the expressed hFVIII maintains a biological activity in plasma from about 50%to about 80%post-dosing. In some instances, the expressed hFVIII maintains a biological activity in plasma from about 25%to about 70%post-dosing. The expressed hFVIII can maintain a biological activity in plasma from about 25%to about 150%about one month after initial dosing. In some instances, the expressed hFVIII maintains a biological activity in plasma from about 50%to about 150%about one month after initial dosing. In some instances, the expressed hFVIII maintains a biological activity in plasma from about 50%to about 100%about one month after initial dosing. In some instances, the expressed hFVIII maintains a biological activity in plasma from about 50%to about 80%about one month after initial dosing. In some instances, the expressed hFVIII maintains a biological activity in plasma from about 25%to about 70%about one month after initial dosing.

PHARMACEUTICAL COMPOSITION

In one aspect, provided herein is a pharmaceutical composition comprising the described rAAV particle or the described composition. In some embodiments, the pharmaceutical composition comprises the rAAV particles of the present disclosure and a pharmaceutically acceptable carrier or excipient.

As used herein, “pharmaceutically or therapeutically acceptable carrier or excipient” refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host or patient. The type of carrier used in the pharmaceutical formulation will depend on the method of administration of the therapeutic compound. Many methods of preparing pharmaceutical compositions for multiple routes of administration are well known in the art.

In some embodiments, the pharmaceutical composition is prepared by dissolving the rAAV virus particles of the present disclosure in a suitable solvent. Suitable solvents include, but are not limited to, water, saline solutions (e.g., NaCl) , buffer solutions (e.g., phosphate-buffered saline (PBS) ) , or other solvents. In certain embodiments, the viral particle pharmaceutical composition may include a surfactant (e.g., Poloxamer, pluronic acid F68) . In certain embodiments, the solvent is sterile. In certain embodiments, the viral particle pharmaceutical composition comprises sodium chloride, sodium phosphate and poloxamer. In some embodiments, the pharmaceutical composition does not comprise any preservatives.

In some embodiments, the pharmaceutical composition is a suspension. In some embodiments, the pharmaceutical composition is a solution.

A pharmaceutical composition described herein can comprise any suitable amount of rAAV particles. In some embodiments, the pharmaceutical composition comprises 1x10^9 to 1x10^15 vector genomes (vg) per mL. In some embodiments, the pharmaceutical composition comprises 1x10^10 to 1x10^14 vg per mL. In some embodiments, the pharmaceutical composition comprises 5x10^10 to 5x10^13 vg per mL. In some embodiments, the pharmaceutical composition comprises 1x10^11 to 1x10^13 vg per mL. In some embodiments, the pharmaceutical composition comprises 0.1 to 500 mL in volume. In some embodiments, the pharmaceutical composition comprises 0.2 to 50 mL in volume. In some embodiments, the pharmaceutical composition comprises 0.1 to 10 mL in volume.

The pharmaceutical composition disclosed herein may also be combined with other therapeutic agents. In some embodiments, the additional therapeutic agent comprises emicizumab

FVIII Fc-fusion protein (rFVIII-Fc;

) , or PEGylated, full- length, recombinant FVIII peptides.

TREATMENT METHOD

The present disclosure also provides a method for treating hemophilia A, which comprises administering a therapeutically effective amount of the polynucleotide, the rAAV, or the pharmaceutical composition of the present disclosure to a subject in need thereof.

In some embodiments, the present disclosure provides methods for treating hemophilia A, which comprises administering to a subject in need of an rAAV particle or a pharmaceutical composition comprising a nucleic acid sequence encoding FVIII at an effective dose and an administration interval such that at least one symptom or feature of hemophilia A is reduced in intensity, severity, or frequency or has delayed in onset.

In some embodiments, the present disclosure provides methods and compositions for delivering a DNA sequence encoding FVIII to a subject for the treatment of hemophilia A. A suitable FVIII DNA sequence encodes any full length, fragment, portion or variant of a FVIII protein which can be substituted for naturally-occurring FVIII protein activity and/or reduce the intensity, severity, and/or frequency of one or more symptoms associated with hemophilia A.

In some embodiments, the system comprising the rAAV particles is provided in a therapeutically effective amount that achieves the desired biological effect at a medically acceptable level of toxicity. The dosage can vary according to the route of administration and the severity of the disease. The dosage can also be adjusted according to the weight, age, sex, degree of symptoms of each patient to be treated, or any combinations thereof. The precise dosage and route of administration can ultimately be determined by the treating doctor or veterinarian. Understandably, the dosage may need to be routinely changed according to the age and weight of the patient and the severity of the condition to be treated.

In some embodiments, the therapeutically effective amount is generally about 1×10^8 to 1×10^18 rAAV virus particles. In some embodiments, the therapeutically effective amount is generally about 1×10^9 to 1×10^17 rAAV virus particles. In some embodiments, the therapeutically effective amount is generally about 1×10^10 to 1×10^17 rAAV virus particles. In some embodiments, the therapeutically effective amount is generally about 1×10^11 to 1×10^17 rAAV virus particles. In some embodiments, the therapeutically effective amount is generally about 1×10^12 to 1×10^17 rAAV virus particles. In some embodiments, the therapeutically effective amount is generally about 1×10^13 to 1×10^17 rAAV virus particles. In some embodiments, the therapeutically effective amount is generally about 1×10^14 to 1×10^17 rAAV virus particles. In some embodiments, the therapeutically effective amount is generally about 1×10^15 to 1×10^17 rAAV virus particles. In some embodiments, the therapeutically effective amount is generally about 1×10^16 to 1×10^17 rAAV virus particles.

In some embodiments, the frequency of administration may be applied at least once a day, including 2, 3, 4, or 5 times a day. In some embodiments, the treatment can last for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 150 days, 200 days, 250 days, 300 days, 400 days, 500 days, 750 days, 1000 days, or more than 1000 days.

Provided methods of the present disclosure contemplate single as well as multiple administrations of a therapeutically effective amount of the therapeutic agents (e.g., rAAV comprising a DNA sequence encoding a FVIII protein) described herein. Therapeutic agents can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition (e.g., hemophilia A) . In some embodiments, a therapeutically effective amount of the therapeutic agents (e.g., rAAV comprising a nucleic acid sequence encoding a FVIII protein) described here may be administered intravascularly periodically at regular intervals (e.g., once every year, once every six months, once every five months, once every three months, bimonthly (e.g., once every two months) , monthly (e.g., once every month) , biweekly (e.g., once every two weeks, every other week) , weekly, daily or continuously) .

In some embodiments, provided rAAV and/or compositions comprising rAAV are formulated such that they are suitable for extended release of the nucleic acid encoding a FVIII protein contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in some embodiments, the compositions of the present disclosure are administered to a subject twice day, daily or every other day. In some embodiments, the compositions of the present disclosure are administered to a subject twice a week, once a week, every 7 days, every 10 days, every 14 days, every 28 days, every 30 days, every two weeks (e.g., every other week) , every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually. Also contemplated are compositions which are formulated for depot administration (e.g., intramuscularly, subcutaneously) to either deliver or release rAAV over extended periods of time.

In some embodiments, a therapeutically effective amount of the composition can be determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present disclosure. As disclosed above, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, modulating, curing, preventing and/or ameliorating hemophilia A) . For example, a therapeutically effective amount can be an amount sufficient to achieve a desired therapeutic and/or prophylactic effect. In some cases, the amount of a therapeutic agent (e.g., rAAV comprising a DNA sequence encoding a FVIII protein) administered to a subject in need thereof depends upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. In some cases, both objective and subjective assays can optionally be employed to identify optimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein or therapeutic agent or therapeutic composition, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) can vary, for example, depending on route of administration, on combination with other pharmaceutical agents. In some cases, the specific therapeutically effective amount (and/or unit dose) for any particular patient can depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific protein employed; the duration of the treatment; etc.

In some cases, a therapeutically effective dose of the provided composition, when administered regularly, results in increased expression of hepatic FVIII protein as compared to baseline levels before treatment. In some embodiments, administering the provided composition results in the expression of a FVIII protein level at or above about 100 ng/ml, about 200 ng/ml, about 300 ng/ml, about 400 ng/ml, about 500 ng/ml, about 600 ng/ml, about 700 ng/ml, about 800 ng/ml, about 900 ng/ml, about 1,000 ng/ml, about 1, 200 ng/ml or about 1, 400 ng/ml of total protein in serum.

In some embodiments, administering provided compositions results in increased serum FVIII protein levels. In some embodiments, administering provided compositions results in increased serum FVIII protein levels by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%as compared to baseline FVIII protein level before treatment. Baseline FVIII protein level in serum is measured immediately before or at a commonly selected time before the treatment.

In some embodiments, administering the provided composition results in increased circulating FVIII levels in a biological sample. Suitable biological samples include, for example, whole blood, plasma, and serum. In some embodiments, administering the provided composition results in increase of circulating FVIII levels in a biological sample (e.g., a serum, or plasma) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%as compared to baseline level before treatment. The baseline circulating FVIII level is measured immediately before or at a commonly selected time before the treatment.

In some embodiments, the composition is administered by intravascular injection, intravenous injection, or intra-arterial injection.

In some embodiments, a therapeutically effective dose of the provided composition, when administered regularly, results in an increased circulating FVIII level in serum or plasma as compared to the baseline circulating FVIII level immediately before or at a commonly selected time before the treatment. In some embodiments, a therapeutically effective dose of the provided composition, when administered regularly, results in an increased circulating FVIII level in serum or plasma as compared to the baseline FVIII level in subjects who are not treated. In some embodiments, a therapeutically effective dose of the provided composition, when administered regularly, results in increase of circulating FVIII levels to about 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml., 90 ng/ml, 100 ng/ml, 110 ng/ml, 120 ng/ml, 130 ng/ml, 140 ng/ml, 150 ng/ml, 160 ng/ml, 170 ng/ml, 180 ng/ml, 190 ng/ml, 200 ng/ml in serum or plasma.

In some embodiments, the therapeutically effective dose ranges from about 1 × 10 ¹⁰ vg/kg to 1 × 10 ¹⁵ vg/kg body weight, e.g., from about 1 × 10 ¹⁰ vg/kg to 2 × 10 ¹¹ vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 4 × 10 ¹¹ body weight, from about 1 × 10 ¹⁰ vg/kg to 8 × 10 ¹¹ vg/kg body weight from about 1 × 10 ¹⁰ vg/kg to 2 × 10 ¹² vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 4 × 10 ¹² vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 8 × 10 ¹² vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 2 × 10 ¹³ vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 4 × 10 ¹³ vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 8 × 10 ¹³ vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 2 × 10 ¹⁴ vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 4 × 10 ¹⁴ vg/kg body weight, from about 1 × 10 ¹⁰ vg/kg to 8 × 10 ¹⁴ vg/kg body weight. In some embodiments, the DNA sequence coding FVIII is administered at a dose ranging from about 0.1-5.0 mg/kg body weight, for example about 0.1-4.5, 0.1-4.0, 0.1-3.5, 0.1-3.0, 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3, 0.3-5.0, 0.3-4.5, 0.3-4.0, 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, 0.3-0.5, 0.5-5.0, 0.5-4.5, 0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, or 0.5-1.0 mg/kg body weight.

In some embodiments, the therapeutically effective dose is or greater than about 1 ×10 ¹⁰ vg/kg body weight, about 2 × 10 ¹⁰ vg/kg body weight, about 4 × 10 ¹⁰ vg/kg body weight, about 6 × 10 ¹⁰ vg/kg body weight, about 8 × 10 ¹⁰ vg/kg body weight, about 1 × 10 ¹¹ vg/kg body weight, about 2 × 10 ¹¹ vg/kg body weight, about 4 × 10 ¹¹ vg/kg body weight, about 6 × 10 ¹¹ vg/kg body weight, about 8 × 10 ¹¹ vg/kg body weight, about 1 × 10 ¹² vg/kg body weight, about 2 × 10 ¹² vg/kg body weight, about 4 × 10 ¹² vg/kg body weight, about 6 × 10 ¹² vg/kg body weight, about 8 × 10 ¹² vg/kg body weight, about 1 × 10 ¹³ vg/kg body weight, about 2 × 10 ¹³ vg/kg body weight, about 4 × 10 ¹³ vg/kg body weight, about 6 × 10 ¹³ vg/kg body weight, about 8 × 10 ¹³ vg/kg body weight, about 1 × 10 ¹⁴ vg/kg body weight, about 2 × 10 ¹⁴ vg/kg body weight, about 4 × 10 ¹⁴ vg/kg body weight, about 6 × 10 ¹⁴ vg/kg body weight, about 8 × 10 ¹⁴ vg/kg body weight, or about 1 × 10 ¹⁵ vg/kg body weight. In some embodiments, the therapeutically effective dose is administered at a dose of or less than about 1 × 10 ¹⁵ vg/kg, 8 × 10 ¹⁴ vg/kg, 6 × 10 ¹⁴ vg/kg, 4 × 10 ¹⁴ vg/kg, 2 × 10 ¹⁴ vg/kg, 8 × 10 ¹³ vg/kg, 6 × 10 ¹³ vg/kg, 4 × 10 ¹³ vg/kg, 2 × 10 ¹³ vg/kg, 8 × 10 ¹² vg/kg, 6 × 10 ¹² vg/kg, 4 × 10 ¹⁴ vg/kg, 2 × 10 ¹² vg/kg, 8 × 10 ¹¹ vg/kg, 6 × 10 ¹¹ vg/kg, 4 × 10 ¹¹ vg/kg, 2 × 10 ¹¹ vg/kg, 8 × 10 ¹⁰ vg/kg, 6 × 10 ¹⁰ vg/kg, 4 × 10 ¹⁰ vg/kg, or 2 × 10 ¹⁰ vg/kg body weight.

REAGENT TEST KIT

In another aspect, the present disclosure provides kits for treating A-type hemophilia, comprising a polynucleotide, an AAV viral particle, or a pharmaceutical composition thereof as disclosed herein, and an instruction manual. In some embodiments, the instructions indicate the administration of the polynucleotide, the AAV viral particle, or the pharmaceutical composition for the treatment of A-type of hemophilia.

In some embodiments, the kit further comprises a container. In some embodiments, the container is configured to deliver the polynucleotides, the AAV viral particle, or the pharmaceutical composition. In some embodiments, the container comprises a vial, a dropper, bottles, tubes, and syringes. In some of the embodiments, the container is a syringe used for administering the polynucleotide, the AAV viral particles, or the pharmaceutical composition.

EXAMPLES

The following examples further illustrate the invention. These examples are only intended to illustrate the invention, and should not be construed as limiting the present invention.

EXAMPLE 1. DESIGN AND CLONING OF RECOMBINANT AAV VECTOR

The rep and cap coding sequences derived from AAV2, AAV5, and/or KP1, respectively, together with their corresponding promoters were synthesized and cloned into modified pFastBac1 to obtain the first polynucleotide comprising the coding sequences of cap and rep proteins.

The nucleotide sequences encoding the FVIII polypeptide variants shown in Table 3 and their corresponding promoters and poly (A) tails were cloned into modified pFastBac1 to obtain the second polynucleotide comprising the coding sequence of FVIII variants. Codon optimization was used to optimize the expression of FVIII and reduce the number of CpG counts.

As shown in FIG. 1, protein expression levels of FVIII encoded by different constructs as disclosed in Table 1 are illustrated. The different construct plasmids were transfected into Huh-7 hepatocellular carcinoma cell line to allow the expression of FVIII. The levels of FVIII in the culture supernatants were measured using the Factor VIII Antigen PLUS ELISA Kit (F8PLUS-AG, Affinity Biologicals) . FIG. 2 demonstrated activity of FVIII encoded by various constructs disclosed herein. The activities of these secreted FVIII proteins were measured using FVIII chromogenic activity assay (BIOPHEN ^TM FVIII: C, 221402-RUO) . FIG. 3 demonstrated specific activity of FVIII encoded by various conducts disclosed herein. The specific activities were obtained by dividing the activities by their corresponding total FVIII protein levels.

The obtained second polynucleotide comprising the coding sequence of FVIII variants were packed into KP1 capsid by using the Bac/Sf9 system to generate AAV vectors. Table 4 provides the information of the AAV vectors comprising corresponding polynucleotides.

Table 4.

Plasmid ID	AAV Vector ID
PA035	AAV #	1
PA036	AAV #	2
PA039	AAV #	3
PA040	AAV #	4
PA042	AAV #	5
PA045	AAV #	6
PA046	AAV #	7
PA048	AAV #	8
PA050	AAV #	9
PA051	AAV #	10

EXAMPLE 2. IN VIVO ACTIVITY OF FVIII IN KNOCKOUT MOUSE MODELS RECEIVING THE AAV VECTORS

The obtained AAV vectors were injected intravenously into FVIII knockout (FVIII-KO) hemophilia A model mice at dose of 4× 10 ¹² vg/Kg. Human FVIII concentration in the mouse plasma was determined using the Factor VIII Antigen PLUS ELISA Kit (F8PLUS-AG, Affinity Biologicals) . The activity of the expressed human FVIII in the mouse plasma was determined by chromogenic activity assay (Chromogenix

SP4 Factor VIII, 82409463) . FIG. 4 shows the human FVIII concentrations in the mouse plasma on day 14 since the initial dosing of various AAV vectors in the FVIII-KO mice. The concentration was measured using the Factor VIII ELISA Kit and calculated as percentage of normal. Data are presented as mean with SEM (n= 7-10; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001 compared with the vehicle control) . FIG. 5 shows the human FVIII activity in the mouse plasma on day 14 since the initial dosing of various AAV vectors in the FVIII-KO mice. The activity of FVIII in mouse plasma was measured by chromogenic activity assay (Chromogenix

SP4 Factor VIII, 82409463) and calculated as percentage of normal. Data are presented as mean with SEM (n= 7-10; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001 compared with the vehicle control) . FIG. 6 shows the human FVIII concentration in the mouse plasma on day 30 since the initial dosing of various AAV vectors in the FVIII-KO mice. Data are presented as mean with SEM (n= 7-10; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001 compared with the vehicle control) . FIG. 7 shows the human FVIII activity in the mouse plasma on day 30 since the initial dosing of various AAV vectors in the FVIII-KO mice. Data are presented as mean with SEM (n= 7-10; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001 compared with the vehicle control) .

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A polynucleotide comprising a nucleic acid encoding a human Factor VIII peptide, wherein the polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 5-22; wherein the Factor VIII peptide lacks a B domain.
A polynucleotide comprising a nucleic acid encoding a human Factor VIII peptide, wherein the polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 22; wherein the Factor VIII peptide lacks a B domain.
The polynucleotide of claim 2, wherein the polynucleotide comprises a sequence of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, or 22.
The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to SEQ ID NOs: 6, 10, 18, or 20.
The polynucleotide of claim 4, wherein said polynucleotide comprises a sequence of SEQ ID NOs: 6, 10, 18, or 20.
The polynucleotide of any one of claims 1-5, wherein said polynucleotide is a recombinant polynucleotide.
The polynucleotide of any one of claims 1-5, wherein said polynucleotide is a synthetic polynucleotide.
The polynucleotide of any one of claims 1-5, wherein said polynucleotide is a combination of a recombinant polynucleotide and a synthetic polynucleotide.
The polynucleotide of any one of the previous claims, wherein said polynucleotide is codon optimized.
The polynucleotide of any one of claims 1-9, wherein said nucleic acid encoding the human Factor VIII peptide comprises a reduced number of CpG dinucleotides as compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain.
The polynucleotide of claim 10, wherein said nucleic acid encoding the human Factor VIII peptide comprises less than about 5 CpG dinucleotides.
The polynucleotide of claim 10, wherein said nucleic acid encoding the human Factor VIII peptide does not comprise a CpG dinucleotide.
The polynucleotide of any one of claim 1-12, wherein said nucleic acid encoding the human Factor VIII peptide has a reduced immunogenicity compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain.
The polynucleotide of claim 4 or claim 5, wherein an expression level of said human Factor VIII peptide is increased by at least about 10%compared to an expression level of a wild-type Factor VIII peptide without a B domain encoded by a wild-type nucleic acid.
The polynucleotide of claim 14, wherein said increased expression level of said human Factor VIII peptide activates a less unfolded protein response (UPR) by at least 5%compared to an expression level of a wild-type Factor VIII peptide without a B domain encoded by a wild-type nucleic acid.
The polynucleotide of claim 14, wherein said increased expression level of said human Factor VIII peptide reduces an endoplasmic reticulum (ER) stress response by at least 5%compared to an expression level of a wild-type Factor VIII peptide without a B domain encoded by a wild-type nucleic acid.
The polynucleotide of any one of claims 1-16, wherein the polynucleotide comprises a promoter operably connected to the nucleic acid encoding the human Factor VIII peptide.
The polynucleotide of claim 17, wherein said promoter is a liver-specific promoter.
The polynucleotide of claim 18, wherein said liver-specific promoter is a transthyretin (TTR) promoter.
The polynucleotide of any one of claims 1-19, wherein said polynucleotide is deoxyribonucleic acid (DNA) .
The polynucleotide of any one of claims 1-19, wherein said polynucleotide is ribonucleic acid (RNA) .
A polynucleotide comprising a nucleic acid encoding a human Factor VIII peptide, wherein said polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, and 22; wherein said Factor VIII peptide lacks a B domain.
The polynucleotide of claim 22, wherein said polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, and 22.
The polynucleotide of claim 22, wherein said polynucleotide comprises a sequence with at least about 90%, 95%, or 99%sequence identity to SEQ ID NOs: 17, 18, 19, 20, 21, or 22.
The polynucleotide of claim 24, wherein said polynucleotide comprises a sequence of SEQ ID NOs: 17, 18, 19, 20, 21, or 22.
A recombinant adeno-associated virus (rAAV) particle, comprising a polynucleotide comprising a nucleic acid encoding a human Factor VIII peptide lacking a B domain, wherein said Factor VIII peptide comprises a F309S mutation; and wherein said rAAV particle comprises an AAV capsid protein selected from the group consisting of AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAVHSC, AAVDJ, LK03, NP59, and KP1.
The rAAV particle of claim 26, wherein the rAAV particle is a single-stranded AAV (ssAAV) .
The rAAV particle of claim 26, wherein said AAV capsid protein is AAV5 or AAV8.
The rAAV particle of any one of claims 26-28, wherein said polynucleotide comprises a sequence with at least about 90%, about 95%, or about 99%sequence identify to SEQ ID NOs: 6, 10, 18, or 20.
The rAAV particle of any one of claims 26-29, wherein said polynucleotide comprises a sequence of SEQ ID NOs: 6, 10, 18, or 20.
The rAAV particle of any one of claims 26-30, wherein said polynucleotide is codon optimized.
The rAAV particle of any one of claims 26-31, wherein said nucleic acid encoding the human Factor VIII peptide comprises less than about 5 CpG dinucleotides.
The rAAV particle of any one of claims 26-32, wherein said nucleic acid encoding the human Factor VIII peptide does not comprise a CpG dinucleotide.
A recombinant adeno-associated virus (rAAV) particle, prepared by transfecting the composition of any one of claims 26-33 into a host cell.
The rAAV particle of claim 34, wherein the host cell is an insect cell, a human cell, or an animal cell.
The rAAV particle of claim 35, wherein the insect cell is a Drosophila S2 cell or a Sf9 cell.
The rAAV particle of claim 36, wherein the animal cell is a fibroblasts, a Chinese hamster ovary (CHO) cell, a COS cell, a murine myeloma (NS0) cell, a HeLa cell, or a Baby Hamster Kidney (BHK) cell.
The rAAV particle of claim 37, wherein the human cell is a human embryonic kidney 293 (HEK293) cell, a HEK293 derivative (such as 293T) , a human fibrosarcoma (HT-1080) cell, a differentiated hepatocyte-derived carcinoma (Huh-7) cell, or a PER. C6 cell.
A pharmaceutical composition, comprising:

(a) the recombinant nucleic acid of claims 1-25 or the recombinant adeno-associated virus (rAAV) particle of claims 26-38; and

(b) a pharmaceutically acceptable excipient.
The pharmaceutical composition of claim 39, wherein the pharmaceutically acceptable excipient comprises serum free media, a lipid, a nanoparticle, vitamin K, surfactant, or phosphate buffered saline (PBS) .
The pharmaceutical composition of claim 40, wherein said surfactant is Pluronic acid F-68.
A method for treating hemophilia A, comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 39-41 to a subject in need thereof.
The method of claim 42, wherein said pharmaceutical composition is administered via intravenous injection.
The method of claim 43, wherein said pharmaceutical composition is administered at a dose of about 10 ⁸ vg/kg -10 ¹⁵ vg/kg.