Recombinant human factor VIII with improved secretory and clotting activity obtained by genetic engineering

Abstract

The present invention relates to engineered human factor VIII (hFVIII) polypeptides that contain at least two substituted amino acids in the A1 domain of hFVIII. The present invention also relates to nucleic acid fragments encoding the engineered hFVIII polypeptides, expression vectors or rAAV vectors that contain such nucleic acid fragments, and methods of treating patients with hemophilia A with the engineered hFVIII polypeptides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to patent application PCT Application No. PCT / CN2021 / 133004, filed November 25, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Human factor VIII is a protein encoded by the F8 gene located on the X chromosome and is composed of 2351 amino acids. Defects in the F8 gene result in the absence or deficiency of the FVIII protein it encodes. Hemophilia A (HA) is an inherited bleeding disorder caused by a lack of FVIII protein, including a lack of clotting activity caused by defective FVIII production, a lack or deficiency of FVIII secretion, or a lack of clotting activity caused by partial or complete inhibition of FVIII by inhibitors. Due to the lack of FVIII, the blood of hemophilia A patients cannot clot normally to stop bleeding.

[0003] A common method to treat HA is replacement therapy. Concentrated factor VIII is slowly infused or injected into the vein of HA patients. These infusions contribute to replacing missing or reduced FVIII in the patient's body. However, this replacement therapy may produce factor VIII inhibitors due to injection or acquired factor VIII inhibitors, which may lead to failure of this replacement therapy. Summary of the Invention

[0004] The present invention provides engineered human factor VIII polypeptides, nucleic acids encoding FVIII proteins and vectors expressing FVIII proteins, pharmaceutical compositions containing FVIII, and methods of using the same, thereby fulfilling a need in the art, such as for the treatment of hemophilia A.

[0005] First, the present invention provides an engineered human factor VIII (hFVIII) polypeptide comprising at least two substituted amino acids in the A1 domain of hFVIII.

[0006] In some embodiments, the substituted amino acids include the L50 and L152 positions of the A1 domain.

[0007] In some embodiments, the substituted amino acids include L50V and L152P in the A1 domain.

[0008] In some embodiments, the substituted amino acids further comprise one or more of the amino acid substitutions selected from the group consisting of D20, G22, 161, D115, F129, G132, Q139, and L159 of the A1 domain.

[0009] In some embodiments, the substituted amino acids further include one or more of the A1 domains D20S, G22L, I61T, A115E, F129I, G132D, Q139E, and L159F.

[0010] In some embodiments, the substituted amino acids include D20S, L50V, and L152P in the A1 domain.

[0011] In some embodiments, the substituted amino acids include D20S, G22L, L50V, and L152P in the A1 domain.

[0012] In some embodiments, the engineered hFVIII polypeptide comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0013] Second, the present invention provides isolated nucleic acid fragments encoding the engineered hFVIII polypeptides disclosed herein.

[0014] Third, the present invention provides an expression vector comprising a nucleic acid fragment disclosed herein operably linked to a promoter.

[0015] Fourth, the present invention provides recombinant AAV (rAAV) vectors comprising the nucleic acid fragment disclosed herein operably linked to a promoter.

[0016] Fifth, there is provided a pharmaceutical composition comprising an expression vector disclosed herein or an rAAV vector disclosed herein.

[0017] Sixth, the present invention provides a method for treating hemophilia A. The method comprises administering to a patient an effective amount of a pharmaceutical composition disclosed herein.

[0018] The above information, features, and advantages of the present invention are better explained in the following description and appended claims. [Brief description of the drawings]

[0019] The innovations of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention may be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the drawings in which:

[0020] [Figure 1] FIG. 1 is a schematic diagram of human wild-type FVIII and FVIII-SQ.

[0021] [Diagram 2] 2A to 2C show the results of the clotting time of heterozygous FVIII, in which Fig. 2A is a comparison of the activity of hHC and mHC, Fig. 2B is a comparison of the activity of hHC and dHC, and Fig. 2C is a comparison of the activity of hHC and maHC.

[0022] [Diagram 3] Figure 3A shows the hybrid FVIII, M1H2, and H1M2, constructed by combining and pairing the A1 and A2 domains of human and giant bat FVIII. Figure 3B shows the clotting time of various FVIII proteins.

[0023] [Figure 4] It is shown that the A1 domain in the heavy chain can be subdivided into D1 and D2 regions, and the A2 domain can be subdivided into D3 and D4 regions.

[0024] [Diagram 5] Figure 5A shows that the D1 or D4 domain of giant bat FVIII is replaced with the human corresponding structure to construct hybrid giant bat FVIIIs: hD1 and hD4. Figure 5B shows the clotting time of various FVIII proteins.

[0025] [Figure 6] Figure 6A shows that the D1-D4 domains of human factor VIII are replaced with those of the giant bat FVIII counterpart to construct hybrid human factor VIIIs: mD1mD3, mD2, mD3, and mD4. Figure 6B shows the clotting time of the various FVIII proteins.

[0026] [Figure 7] 1 shows an alignment of the D1 region sequences of human and giant bat FVIII.

[0027] [Figure 8] Figure 8A shows the results of ELISA, indicating that the eight amino acid mutations V51L, T62I, E116D, I130F, D133G, E140Q, P153L, and F160L result in a decrease in the expression level of FVIII protein. Figure 8B shows the results of aPTT, indicating that the eight amino acid mutations V51L, T62I, E116D, I130F, D133G, E140Q, P153L, and F160L result in a decrease in coagulation activity.

[0028] [Figure 9] 1 shows the clotting activity of various mutant FVIII. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Particular features of the invention are referred to in the Abstract and Detailed Description above, as well as in the claims that follow. It should be noted that the disclosure of the invention herein includes all possible combinations of such particular features. For example, if a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or in the context of a particular claim, this feature can be used in combination with and / or in the context of other aspects and embodiments of the invention, and of the invention generally.

[0030] Replacement therapy for the treatment of hemophilia A generates inhibitors of injected or acquired FVIII, which may lead to failure of replacement therapy.

[0031] Another method for the treatment of hemophilia A is gene therapy based on rAAV vectors. rAAV vectors can stably express target genes in vivo for a long period of time to achieve therapeutic purposes. The coding region of F8 is 7035 bp long and is divided into six domains: A1, A2, B, A3, C1, and C2 (Figure 1, lower diagram). The size of the rAAV vector, which contains an expression cassette of one therapeutic gene and two ITRs, is approximately 5 kb so that the rAAV vector can be efficiently packaged into the outer shell of adeno-associated virus (AAV).

[0032] Due to the limited AAV packaging capacity, the full-length F8 coding region cannot be packaged completely into AAV. To solve this problem, it is necessary to shorten the coding region of the F8 gene. Previous studies have shown that the B domain (908 aa) of FVIII can be replaced with the SQ domain (14 aa) while maintaining the coagulation activity of FVIII. Such a genetically engineered FVIII is called FVIII-SQ and is divided into six domains: A1, A2, SQ, A3, C1, and C2. The A1, A2, and SQ domains constitute the heavy chain of FVIII-SQ, and A3, C1, and C2 constitute the light chain of FVIII-SQ. The nucleotides encoding FVIII-SQ are 4371 bp (Figure 1, upper panel) and therefore can be packaged completely into AAV.

[0033] However, even though the truncated FVIII expression element is about 5 kb, one of the important limiting factors in rAAV gene therapy for hemophilia A is the inefficient secretion of FVIII, which is probably due to the slow folding process of FVIII in the endoplasmic reticulum. To compensate for this, a large amount of rAAV vector needs to be injected into the body of hemophilia A patients to ensure that sufficient active FVIII is produced. A large amount of rAAV vector injected may cause side effects such as immune reactions in patients.

[0034] Since FVIII is a secretory protein, one strategy is to enhance the secretory activity of FVIII produced by rAAV vectors by modifying amino acids of FVIII in order to reduce the required amount of viral vector to an acceptable level. The more FVIII is secreted, the higher the coagulation activity of FVIII. Research has shown that the secretory ability of porcine FVIII is 10-100 times higher than that of human factor VIII, and the heavy chain of porcine FVIII is advantageous for enhancing the secretory ability of FVIII (Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion. JBC, 279, 6546-6552). Therefore, the inventors of the present application have proposed a theory that the heavy chain of human factor VIII can be modified to enhance its secretory ability for rAAV gene therapy, but are not bound by such theory.

[0035] First, the present invention provides an engineered human factor VIII (hFVIII) polypeptide comprising at least two substituted amino acids in the A1 domain of hFVIII.

[0036] As used herein, "genetic engineering" refers to a modification that changes a protein from its wild-type state to another state, by the manipulation of genetic material, chemical synthesis, or other methods. Engineered FVIII may also be referred to as mutant FVIII, heterozygous FVIII, or FVIII mutant, depending on the context.

[0037] As used herein, "substitution" or "replacement" refers to the replacement of an amino acid, i.e., changing one amino acid in a protein to another amino acid due to a point mutation in the DNA sequence. A "substitution amino acid" is a new amino acid that replaces an existing amino acid.

[0038] As used herein, "domain" refers to a contiguous amino acid sequence characterized by internal amino acid sequence identity of structurally related domains and defined by thrombin proteolytic sites. Figure 1 (bottom) shows human wild-type FVIII, including the A1, A2, B, A3, C1, and C2 domains.

[0039] The A1 domain of human factor VIII has been subdivided into D1 and D2 regions in order to determine the amino acid sequence of a smaller region of the A1 domain that is responsible for enhancing secretory activity.

[0040] The amino acid sequence of the human D1 region is defined in SEQ ID NO:1 as follows: ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKE

[0041] In some embodiments, the substituted amino acids include L50 and L152 of the A1 domain, where L50 means that the leucine (L) at position 50 of SEQ ID NO:1 is substituted with another unspecified amino acid, and L152 means that the leucine (L) at position 152 of SEQ ID NO:1 is substituted with another unspecified amino acid.

[0042] In some embodiments, the substituted amino acids include L50V and L152P in the A1 domain, where L50V means that leucine (L) at position 50 of SEQ ID NO:1 is replaced with valine (V) and L152P means that leucine (L) at position 152 of SEQ ID NO:1 is replaced with proline (P).

[0043] In some embodiments, one or more of the amino acids D20, G22, I61, A115, F129, G132, Q139, and L159 of the A1 domain are substituted.

[0044] In some embodiments, the amino acid substitutions of D20, G22, I61, D115, F129, G132, Q139 and L159 are D20S, G22L, I61T, D115E, F129I, G132D, Q139E and L159F, respectively.

[0045] In some embodiments, the substituted amino acids include D20S, L50V, and L152P.

[0046] In some embodiments, the substituted amino acids include D20S, G22L, L50V, and L152P.

[0047] In some embodiments, the engineered hFVIII polypeptide comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0048] SEQ ID NO:3: ATRRYYLGAVELSWDYMQSSLLELPVDARFPPRVPKSFPFNTSVVYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDL NSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLR

[0049] SEQ ID NO:4: ATRRYYLGAVELSWDYMQSSLGELPVDARFPPRVPKSFPFNTSVVYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDL NSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLR

[0050] SEQ ID NO:5:

[0051] SEQ ID NO:6:

[0052] Second, the present invention provides isolated nucleic acid fragments encoding the engineered hFVIII polypeptides disclosed herein. The isolated nucleic acid fragments include all possible nucleic acid sequences encoding the FVIII mutants described herein. All possible nucleic acid sequences take into account, but are not limited to, the principle of codon degeneracy.

[0053] Third, the present invention provides an expression vector comprising a nucleic acid fragment disclosed herein operably linked to a promoter.

[0054] The term "operably linked" means that regulatory sequences required for expression of a coding sequence are positioned in the DNA molecule in the appropriate position to allow expression of the coding sequence.

[0055] Fourth, the present invention provides recombinant AAV (rAAV) vectors comprising the nucleic acid fragment disclosed herein operably linked to a promoter.

[0056] Human adeno-associated virus (AAV) is a non-pathogenic virus that replicates efficiently only in cells co-infected with a helper virus (usually adenovirus or herpesvirus). The virus has a broad host range and can effectively infect many cell types in different species. However, no studies have yet demonstrated the association of AAV with human or animal disease.

[0057] AAV binds to cells via heparin sulfate proteoglycan receptors. Once attached, AAV entry into the cell is dependent on receptors, namely fibroblast growth factor receptor and αvβ5 integrin molecules. AAV enters the infected cell in the form of single-stranded DNA (ssDNA) and is further converted into a double-stranded transcription template. Cells infected with AAV and a helper virus undergo AAV replication induced by the helper virus, not AAV itself, before the cell divides. Proteins or RNA transcripts encoded by the helper virus are transcription regulators, involved in DNA replication or modifying the cellular environment, thereby ensuring efficient production of the virus.

[0058] Recombinant AAV (rAAV) vectors typically replace viral coding sequences with a gene of interest. These vectors have been proven to have efficient expression and gene targeting at several different sites in vitro and in vivo. Studies have shown that AAV is safe and allows stable and sustained expression in respiratory, central nervous system, skeletal muscle, liver and eye studies. As the titer and purity of rAAV preparations have improved, so has the efficiency of rAAV-mediated transduction.

[0059] The inverted terminal repeats (ITRs) of the AAV genome are the only cis elements required for the synthesis of rAAV vectors. The target gene and expression elements are added to the two ITRs to create a ssDNA vector genome of approximately 5 kb in size, which is then packaged into AAV particles in the presence of the AAV rep and cap genes and a helper virus, and methods and technologies for the production and purification of mature rAAV are available.

[0060] In the rAAV vector, the nucleic acid fragment encoding the engineered FVIII described herein is less than 5 kb and is inserted in expression frame containing two ITRs to allow efficient packaging of the AAV vector.

[0061] In the expression vector or rAAV vector, the nucleic acid sequence encoding the engineered factor VIII disclosed herein is operably linked to a promoter, which may be, but is not limited to, a constitutive promoter, an inducible promoter, a liver-specific promoter, a hepatocyte-specific promoter, or a synthetic promoter.

[0062] The constitutive promoter may be, but is not limited to, a herpes simplex virus (HSV) promoter, a thymidine kinase (TK) promoter, a Rous sarcoma virus (RSV) promoter, a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, an adenovirus E1A promoter, a cytomegalovirus (CMV) promoter, a mammalian housekeeping gene promoter, or a β-actin promoter.

[0063] The inducible promoter may be, but is not limited to, a cytochrome P450 gene promoter, a heat shock protein gene promoter, a metallothionein gene promoter, a hormone-inducible gene promoter, an estrogen gene promoter, or a tetracycline-responsive tetVP16 promoter.

[0064] The liver-specific promoter may be, but is not limited to, an albumin promoter, an alpha-1-antitrypsin promoter, or a Hepatitis B virus core protein promoter.

[0065] A synthetic promoter can include regions of known promoters, regulatory elements, transcription factor binding sites, enhancing elements, inhibitory elements, etc. For example, a synthetic promoter can be a combination of a natural promoter and an enhancer derived from a transcription factor. Any promoter suitable for expression in a target host cell can be used to achieve the goal of increasing the expression level of a target nucleic acid, protein, or polypeptide.

[0066] Fifth, the present invention provides a pharmaceutical composition comprising an expression vector disclosed herein or an rAAV vector disclosed herein.

[0067] The term "pharmaceutical composition" refers to a mixture of an expression vector disclosed herein or a rAAV vector disclosed herein with other chemical components, such as diluents or carriers. Pharmaceutical compositions are advantageous for targeting biological tissues. Pharmaceutical compositions are generally tailored according to a particular intended route of administration. Pharmaceutical compositions are suitable for human and / or veterinary use.

[0068] The pharmaceutical compositions described herein can be administered to a human patient by themselves, or in pharmaceutical compositions mixed with other active ingredients, such as in combination therapy, or mixed with carriers, diluents, excipients, or combinations thereof. The exact formulation will depend on the route of administration chosen.

[0069] Sixth, the present invention provides a method for treating a patient with hemophilia A. The method comprises administering to the patient an effective amount of the pharmaceutical composition disclosed in the present invention.

[0070] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition, such as a composition comprising an rAAV vector, that should ensure the expected activity when injected into a desired subject. The term "therapeutically effective" means sufficient to alleviate the manifestation of, arrest the progression of, or alleviate or relieve at least one of the symptoms of the disease being treated by the disclosed method.

[0071] As one of skill in the art will recognize, a treatment can also be considered therapeutically "effective" if the disorder or its symptoms and / or effects are partially ameliorated or alleviated in a subject, even if the disorder is not completely eradicated or prevented. Various indicators for determining the effectiveness of a method for treating a patient with hemophilia A are known to those of skill in the art.

[0072] As used herein, the terms "treating," "treatment," "therapeutic," or "therapy" do not necessarily mean a complete cure or elimination of a disease or condition. Any degree of alleviation of a disease or condition may be considered treatment and / or therapy. Additionally, treatment may include actions that may improve or worsen a patient's overall sense of well-being.

[0073] Other ingredients, such as other active agents, preservatives, buffers, salts, pharma- ceutically acceptable carriers, or other pharma- ceutically acceptable ingredients, may be included in the desired composition.

[0074] As used herein, "solvent" refers to a compound that is advantageous for introducing a compound into cells or tissues. For example, and unless otherwise limited, dimethylsulfoxide (DMSO), ethanol (EtOH) or PEG400 are common solvents that are advantageous for the absorption of organic compounds into cells or tissues of a subject.

[0075] definition 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. In the event that there are multiple definitions for any of the terms herein, those in this section prevail unless otherwise stated.

[0076] Terms used herein are used only to describe a particular situation and are not meant to be limiting. As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. Furthermore, when terms such as "including," "includes," "having," and "has" are used in the detailed description and / or claims, these terms are intended to indicate that similar terms to "comprising" are inclusive.

[0077] As used herein, the terms "individual," "patient," or "subject" may be used interchangeably. These terms do not require or limit the identity (e.g., continuous or intermittent) of the medical practitioner (e.g., physician, licensed practical nurse, licensed nurse associate, physician assistant, nurse, hospice worker).

[0078] Isolated: An "isolated" biological component (e.g., a nucleic acid molecule, a protein, a virus, or a cell) has been isolated or purified from other biological components in the cells or tissues of an organism or organism in which it exists in nature, e.g., other chromosomal and extrachromosomal DNA, RNA, proteins, cells, etc. "Isolated" nucleic acid molecules and proteins include those that have been purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0079] Recombinant: A recombinant nucleic acid molecule is a nucleic acid molecule having a sequence that is not naturally occurring, e.g., contains one or more nucleic acid substitutions, deletions, or insertions, and / or has a sequence that has been created by the artificial combination of two fragments of sequences that are originally isolated. Such an artificial combination can be achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated nucleic acid fragments, e.g., by genetic engineering techniques. This can be done by:

[0080] The term "promoter region" or "promoter" refers to a DNA region that induces / initiats transcription of a nucleic acid (e.g., a gene). Promoters include necessary nucleic acid sequences near the transcription start site. Usually, promoters are located close to the genes they transcribe. Promoters can also optionally contain distal enhancer or suppressor elements that can be located as far away as several thousand base pairs from the transcription start point. A tissue-specific promoter is a promoter that induces / initiats transcription primarily in a single type of tissue or cell. For example, a liver-specific promoter refers to a promoter that is much more effective at directing / initiat-ing transcription in liver tissue than in other tissue types.

[0081] Enhancer: A nucleic acid sequence that increases the activity of a promoter, thereby increasing the efficiency of transcription.

[0082] The term "vector" refers to a small vector DNA molecule into which a DNA sequence can be inserted and replicated in a host cell. An "expression vector" is a special vector that contains a gene or nucleic acid sequence of interest and has the regulatory elements necessary for its expression in a host cell.

[0083] The term "operably linked" means that regulatory sequences necessary for expression of a coding sequence are located in the appropriate location in the DNA molecule to achieve expression of the coding sequence. This definition may also be applied to the sequence of a coding sequence and transcription control elements (such as promoter, enhancer and terminator elements) in an expression vector. This definition may also be applied to the sequence of a mixed nucleic acid molecule sequence generated by a first and a second nucleic acid molecule.

[0084] The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination molecule. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and deoxynucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nuclease-resistant nucleotide derivatives. The term nucleotide as used herein may also refer to dideoxynucleoside triphosphates (ddNTPs) and derivatives thereof. Dideoxynucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled, tagged with a detectable tag by related technology, or labeled with a quantum dot. Detectable tags include radioisotopes, fluorescent tags, chemiluminescent tags, bioluminescent tags, and enzyme tags. Nucleotide fluorescent tags can include, but are not limited to, fluorescein, 5-carboxylfluorescein (FAM), 2,7-dimethoxy-4,5-dichloro-6-carboxylfluorescein (JOE), rhodamine, 6-carboxylrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxylrhodamine (TAMRA), 6-carboxyl-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanide, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G] dUTP, [TAMRA] dUTP, [R110] dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP and [dROX] ddTTP (Perkin Elmer, Foster City, Calif); FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP and FluoroLink Cy5-dUTP (Amersham, Arlington Heights, II); may include fluorescein-15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP and fluorescein-15-2'-dATP (Boehringer Mannheim, Indianapolis, Ind.). Chromosomally labeled nucleotides include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP (Molecular Probes, Eugene, Oreg.).Nucleotides may also be labeled or annotated by chemical modification. The chemically modified mononucleotide may be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs may include biotin-dATP (e.g., biotin-N6-ddATP and biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP and biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, and biotin-20-dUTP).

[0085] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" may be used interchangeably and refer to different polymeric forms of nucleotides, including deoxyribonucleotides, ribonucleotides, and nucleotide analogs, including single-stranded, double-stranded, or multi-stranded forms. Polynucleotides may be exogenous or endogenous. Polynucleotides may be present outside a cell, may be a gene or a fragment thereof, may be DNA or RNA, may have any three-dimensional structure, and may have known or unknown function. Polynucleotides may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases), and modifications to the nucleotide structure may be made before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, heterologous nucleic acid, morpholine, locked nucleic acid, ethylene glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deazaGTP, fluorescent agents (e.g., rhodamine or fluorescein attached to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiuridine, pseudouridine, dihydrouridine, quinine, and biocin. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), small RNAs (microRNAs), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (cell-free DNA (cfDNA) and cell-free RNA (cfRNA)), nucleic acid probes and primers. Non-nucleotide components are embedded in the nucleotide sequence.

[0086] The term "gene" as used herein refers to nucleic acids (e.g., genomic DNA and cDNA) and their corresponding nucleotide sequences involved in transcription of coding RNA. The term genomic DNA referred to herein includes coding regions, non-coding regions, and regulatory elements, as well as 5' and 3' ends. In some uses, the term includes transcribed sequences, 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region includes an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequences (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, genes such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes do not encode a polypeptide. In some cases, the term "gene" includes not only the transcribed sequence, but also the non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. Gene can refer to an "endogenous gene," or a gene present in the genome of an organism itself, or to an "exogenous gene," or a gene not present in the organism itself. Exogenous genes usually refer to genes not present in the host organism, but can be introduced into the host organism by gene transduction. Exogenous genes can also refer to genes not present in the genome of an organism, or changes in a nucleic acid or polypeptide sequence that occur in an organism, including mutations, insertions and / or deletions (e.g., non-native sequences).

[0087] cDNA (complementary DNA): cDNA refers to DNA obtained by reverse transcription of messenger RNA extracted from cells in the laboratory. cDNA can also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule.

[0088] 5' and / or 3': The orientation of a nucleic acid molecule (e.g., DNA or RNA) is divided into a "5' end" and a "3' end". A mononucleotide forms a phosphodiester bond by esterification between the 3'-terminal hydroxyl group and the 5'-terminal phosphate residue of the adjacent pentose ring, thereby becoming a polynucleotide. Thus, when the 5'-terminal phosphate group of a linear polynucleotide is not linked to the 3'-terminal oxygen of the pentose ring of a mononucleotide, it is called a 5' end. When the 3'-terminal oxygen of a polynucleotide is not linked to the 5'-phosphate group of the pentose ring of another mononucleotide, it is called a 3' end. Although the 5'-phosphate group of the pentose ring in a polynucleotide is linked to the adjacent 3' oxygen, each internal nucleic acid is also divided into a 5' end and a 3' end.

[0089] In linear or circular nucleic acid molecules, isolated internal groups are referred to as "upstream" (5' end) or "downstream" (3' end). In the case of DNA, this term indicates that transcription occurs along the 5' to 3' direction of the DNA strand. Promoter and enhancer elements that direct transcription of the relevant gene are usually located at the 5' or upstream end of the coding region. However, enhancer elements can function even when located at the 3' end of the promoter element and coding region. Transcription terminators and polyadenylation (PolyA) signals are located at the 3' or downstream end of the coding region.

[0090] Transcription Factor (TF): A protein that binds to specific DNA sequences and regulates the transfer (or transcription) of genetic information from DNA to RNA. TFs perform this function alone or in complexes with other proteins by enhancing (as activators) or inhibiting (as inhibitors) the recruitment of specific genes by RNA polymerase (the enzyme that transcribes genetic information from DNA to RNA). The specific DNA sequences to which TFs bind are called response elements (REs) or regulatory elements. Other terms include cis-elements and cis-acting transcriptional regulatory elements.

[0091] As used herein, a "corresponding" nucleic acid or amino acid or any one sequence refers to a site present in FVIII or a fragment thereof, whose structure and / or function is identical to a site in a FVIII molecule of another species, even if the number of nucleotides or amino acids is not exactly identical.

[0092] Control: A control is an individual or group of samples used as a standard for comparison of response studies or experimental results. A control group may also be referred to as a reference group.

[0093] As used herein, a "subunit" of human or animal FVIII refers to the heavy and light chains of the FVIII protein. The heavy chain of FVIII contains three domains, namely A1, A2, and B, and the light chain of FVIII also contains three domains, namely A3, C1, and C2.

[0094] As used herein, "FVIII deficiency" includes defective FVIII production, deficient or non-production of FVIII, or lack of clotting activity due to partial or total inhibition of FVIII by an inhibitor. Hemophilia A is a disease in which impaired clotting function occurs due to a defect in a linked gene on the X chromosome and the absence or deficiency of the FVIII protein it encodes.

[0095] As used herein, "diluent" refers to a component in a pharmaceutical composition that has no medicinal effect but may be pharma- ceutical necessary or desirable. For example, a diluent may be used to increase the effective drug volume when the drug mass is too small or impossible to manufacture and / or administer. A diluent may be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is an aqueous buffer solution, including but not limited to phosphate buffer, which mimics human blood components.

[0096] As used herein, "excipient" refers to an inert substance added to a pharmaceutical composition to ensure the composition's volume, consistency, stability, binding ability, lubricity, disintegration ability, etc. A "diluent" is a type of excipient.

[0097] The terms "treatment" and "treating" as used herein refer to a method of obtaining an effective or expected result, including but not limited to effective treatment and / or effective prevention. For example, treatment can include targeting a system or cell population disclosed herein. Therapeutically effective can refer to a therapeutic improvement or effect on one or more diseases, conditions or symptoms. For effective prevention, the pharmaceutical composition can be used on a subject who may be suffering from a particular disease, condition or symptom, or a subject who may not yet be suffering from the disease, condition or symptom, but is suffering from physiological symptoms of one or more diseases.

[0098] A "therapeutic effect" occurs when the condition being treated is altered, and the change may be positive or negative. For example, a "positive effect" may increase the number of activated T cells in a subject, and in another example, a "negative effect" may correspond to a reduction in the number or size of tumors in the subject. A "change" during treatment may refer to at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 25%, 50%, 75%, or 100% change in the disorder. The change may be based on the degree of improvement in the severity of the disorder during treatment of an individual, or the difference in the degree of improvement in the severity of the disorder between treated and untreated populations. Similarly, the disclosed method may measure the amount of cells that produce a "therapeutically effective" effect in a subject. The term "therapeutically effective" is defined as "having a therapeutic effect."

[0099] The following illustrative description is intended to provide those skilled in the art with a complete indication and description of how to make and use the present invention, but is not intended to limit the scope of the inventors' invention, nor is it intended to represent that the following experiments are all or the only experiments performed. Efforts have been made herein to ensure accuracy of numerical values ​​(e.g., numbers, temperatures) used, but some experimental error and deviation should be accounted for. Unless otherwise specified, weight refers to the weight of an object, molecular weight refers to weight average molecular weight, temperature refers to degrees Celsius, and pressure refers to atmospheric pressure or near atmospheric pressure. EXAMPLES

[0100] Methods and Materials Plasmid construction To prepare plasmids containing FVIII heavy chain mutants, plasmid pAAV-CB-FVIII-SQ is used as a vector. This plasmid contains the CB promoter consisting of the cytomegalovirus enhancer and human β-actin promoter, and the FVIII gene encoding human factor VIII-SQ, which is inserted into the heavy chain mutation site by PCR amplification, and the FVIII genes of human and giant bat are used as templates, and primers are synthesized by Omega Bio-Tech Co., Ltd. The vector and the target gene fragment are enzymatically digested with Not I-HF and Kpn I-HF (NEB) restriction enzymes, and then purified with an EZNA gel extraction kit (Omega Bio-Tech). The sticky ends produced by the enzyme digestion are used to ligate the vector and the target gene to prepare plasmids with various heavy chain mutants.

[0101] Tissue culture and transfection HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Gibco), 100 μg penicillin / mL, and 100 U streptomycin / mL. For transfection, 0.75 μg of FVIII plasmid and 2.25 μL of PolyJet (SignaGen Labs) were mixed and added to each well of a 12-well plate according to the manufacturer's instructions. Six hours after transfection, the medium was replaced with Ham's F12 medium (Corning) containing 2% heat-inactivated fetal bovine serum. 24 hours after medium replacement, the supernatant medium was collected and assayed using aPTT (Activated Partial Thromboplastin Time) and ELISA (Enzyme Linked Immunosorbent Assay).

[0102] aPTT In the in vitro test, the standard was Refacto (Genetics Institute, Cambridge, MA), diluted 1 / 512 from 1U / mL (200 ng / mL) with medium, and the detection sample was obtained by centrifuging the supernatant medium taken from the transfected mammalian cells at 13,000 rpm for 1 minute. STA-PTT reagent, FVIII-deficient plasma, and sample (or diluted standard) were mixed in STAGO blood clotting cups in an amount of 50 μL each, incubated at 37°C for 170 seconds, and then the clotting time was recorded. At the same time, 50 μL of 25 mM CaCl2 was added and measured, and the FVIII activity was calculated according to the calibration curve.

[0103] ELISA Refacto was used as the standard, diluted 1 / 512 in half from 1U / mL (200ng / mL) in culture medium. The supernatant medium after plasmid transfection was used as the sample, as described above. Mouse plasma samples were diluted in HEPES buffer at a dilution ratio of 1:10. The capture antibody (PAH-FVIII-S, 7.1mg / mL, 1:2000) was diluted in coating solution (0.1M sodium bicarbonate and carbonate, pH 9.6), then coated on a 96-well plate, incubated overnight at 4°C, blocked with 3% BSA blocking solution, and the blocking solution was diluted in PBST buffer (140mM NaCl, 2.5mM KCl, 8mM Na2HPO4, 2mM KH2PO4, and 0.05% Tween 20, pH 8.4) and incubated at room temperature for 1 hour. After washing three times with PBST buffer, 100 μL of standards and samples were added and incubated at room temperature for 1 h, washed three times with PBST buffer, detection antibody (GMA-8021-HRP, 1:200) was added and incubated at room temperature for 1 h, washed three times with PBST buffer, and color development was performed using 1× SureBlue TMB one-component Millipore peroxidase substrate, and when color development was performed at room temperature for 1-10 min, it was terminated by adding 0.5 M H2SO4. The OD values ​​were measured at 450 nm and 630 nm, respectively, using a spectrophotometer, and the protein content of FVIII was calculated from the calibration curve.

[0104] Example 1: Determination of the secretion-enhancing structure in the FVIII heavy chain A1 domain of giant bat Research has reported that the secretion ability of porcine FVIII is 10 to 100 times higher than that of human factor VIII, and that the heavy chain of porcine FVIII is advantageous in improving the secretion ability (Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion. JBC, 279, 6546-6552). The present inventors hypothesized that FVIII of other animals may also have the ability to enhance secretion, and devised an experiment to determine the heavy chain region that enhances the secretion ability.

[0105] To this end, we sequenced the nucleotide sequences of the F8 genes of various animals. We selected FVIIIs from monkeys, giant bats, and dolphins because monkeys jump on land, giant bats fly in the air, and dolphins swim in the water. We heterozygoused the FVIII heavy chain sequences of these animals with human FVIII light chain sequences to form hybrid FVIII molecules, and then transfected the plasmids containing these hybrid FVIII molecules into cells. 24 hours after transfection, we collected the supernatant medium and performed aPTT tests to measure the clotting activity of the FVIII molecules.

[0106] The results of aPTT are shown in Figures 2A-2C. mHC is a heterozygous FVIII consisting of a heavy chain of giant bat FVIII and a light chain of human factor VIII, and dHC is a heterozygous FVIII consisting of a heavy chain of dolphin FVIII and a light chain of human factor VIII. In Figures 2A-2C, human factor VIII (hHC, referring to FVIII-SQ) is used as a control, and Figure 2A is a comparison of the activity of hHC and mHC, Figure 2B is a comparison of the activity of hHC and dHC, and Figure 2C is a comparison of the activity of hHC and maHC.

[0107] As shown in Figures 2A-2C, heterozygous FVIII (mHC) containing giant bat FVIII heavy chain and human factor VIII light chain has the shortest clotting time, which indicates that its secretion level is the highest and its clotting activity is the highest. The difference between mHC and human factor VIII is that mHC contains giant bat FVIII heavy chain. This result indicates that the giant bat FVIII heavy chain is advantageous for enhancing the secretion ability of heterozygous mHC FVIII.

[0108] Next, we replaced the A1 and A2 domains of human and giant bat FVIII to construct more heterozygous FVIII molecules, M1H2 and H1M2, respectively: M1H2 contains the A1 domain of giant bat heavy chain (mHC) and the A2 domain of human heavy chain (hHC), and H1M2 contains the A1 domain of hHC and the A2 domain of mHC (Figure 3A).

[0109] As shown in Figure 3B, M1H2 has a shorter clotting time than human factor VIII (hHC), indicating its higher secretion level and higher clotting activity. This data suggests that replacement of the A1 domain of mHC with the A1 domain of hHC improves clotting activity.

[0110] Example 2: Determination of regions with low secretion enhancement in the A1 domain To further subdivide the A1 domain, we predicted the heavy chain structure of FVIII using Phymol software, which subdivided the A1 domain of the heavy chain into D1 and D2, and the A2 domain into D3 and D4, which would help to further identify the regions favorable for enhanced secretion of the heavy chain (Figure 4).

[0111] Both negative and positive selection strategies are used. In the negative selection strategy, the D1 or D4 domain of giant bat FVIII was replaced with the D1 or D4 domain of human factor VIII to construct heterozygous giant bat FVIII: hD1 and hD4 (Figure 5A). As shown in Figure 5A, hD1 contains the D1 region of human factor VIII heavy chain (hHC) and the D2-D4 region of giant bat FVIII heavy chain, and hD4 contains the D4 region of human factor VIII heavy chain (hHC) and the D1-D3 region of giant bat FVIII heavy chain. All FVIII mutants contain the same light chain and other necessary regulatory elements, and the same domains or expression elements are not shown in the figure. Human FVIII has a low secretion activity, which reduces the secretion efficiency of the heterozygous giant bat FVIII mutants. In fact, hD1 has a longer clotting time compared to human factor VIII (hHC), as shown in Figure 5B. These results suggest that the D1 domain of human factor VIII may reduce the secretory activity of giant bat FVIII, which in turn reduces its blood coagulation activity. Thus, the D1 domain may be important for FVIII secretion.

[0112] In the positive selection strategy, the D1-D4 regions of human factor VIII were replaced with the corresponding regions of giant bat FVIII to construct heterozygous human factor VIII mutants mD1mD3, mD2, mD3, and mD4 (Figure 6A). Because giant bat FVIII has higher secretion activity, this mutation enhances the secretion efficiency of heterozygous human factor VIIIs. As shown in Figure 6B, the clotting times of all hybrid human factor VIII mutants are shorter than that of human factor VIII (hHC), among which mD1mD3 has the shortest clotting time. This result suggests that the D1 domain may be a critical region for FVIII secretion.

[0113] The results of negative and positive selection experiments suggest that the giant bat D1 region may be an important domain that enhances the giant bat FVIIII secretion ability.

[0114] Example 3: Determination of amino acids responsible for enhanced secretion in the D1 domain By aligning the human D1 (SEQ ID NO: 1) and giant bat D1 (SEQ ID NO: 2, see below for the sequence), we found that there are 23 amino acid differences between the two (Figure 7, indicated by asterisks). Note that the giant bat D1 region has one more amino acid than the human D1 region (Figure 7, "-" in the human sequence indicates one amino acid deletion). In the following, giant bat FVIII and human factor VIII mutants with various mutation sites are constructed. The amino acid positions in the human factor VIII mutant refer to SEQ ID NO: 1, and the amino acid positions in the giant bat FVIII mutant refer to SEQ ID NO: 2.

[0115] The amino acid sequence of SEQ ID NO:2 is as follows: ATRRYYLGAVELSWDYMQSELLSELHMDTRFPPEVPRSPFNTSVIYKKTVFVEFTDHLFNTAKPRPPWMGLLGPTIRAEVSDTVVITLKNMASHAVSLHAVGVSYWKASEGAQYEDQTSQREKEDDKVIPGDSHTYVWEVLKENGPMASDPPCLTYSYFSHVDLVKDLNAGLIGTLLVCREGSLAKE

[0116] To determine the amino acids that affect the secretion of FVIII protein, 8 of the 23 different amino acids in giant bat D1 were replaced with the corresponding amino acids in human D1, and the corresponding FVIII mutants were obtained: V51L, T62I, E116D, I130F, D133G, E140Q, P153L, and F160L. V51L refers to the 51st amino acid V in SEQ ID NO:2 of the giant bat FVIII mutant mutated to L (see FIG. 7), and the definitions of the other mutants are similar. FIG. 8A shows the results of ELISA, which show that the mutants containing 8 amino acids each, namely V51L, T62I, E116D, I130F, D133G, E140Q, P153L, and F160L, reduce the expression level of FVIII protein (FIG. 8A). Figure 8B shows the aPTT results, which show that the clotting activity of the mutants containing each of the eight amino acids, namely V51L, T62I, E116D, I130F, D133G, E140Q, P153L, and F160L mutants, is reduced (Figure 8B). These data indicate that these eight amino acids play an important role in enhancing the secretion capacity of giant bat FVIII, and the corresponding mutations of the eight amino acids in human factor VIII may enhance the secretion capacity of human factor VIII. Because the D1 region of giant bat FVIII has one more amino acid than human factor VIII (see Figure 7), the human factor VIII mutants L50V, I61T, D115E, F129I, G132D, Q139E, L152P, and L159F may increase the secretion of human factor VIII.

[0117] It should be noted that the secretion of giant bat FVIII was maximally reduced by including two amino acid mutations, V51L and P153L (Figures 8A-8B). In human factor VIII, the L50V and L152P mutations may be two important amino acid mutations that increase the secretion and clotting activity of human factor VIII.

[0118] Example 4: Determination of other amino acids that contribute to enhanced secretory activity At the 20th to 23rd amino acid positions of the D1 region of the giant bat, the amino acid sequence of the giant bat FVIII is ELLS (Figure 7), and the corresponding amino acid sequence of the human factor VIII sequence is DLG (Figure 7). Since FVIII is a secretory protein, it must be released from the synthesis site of the cell and pass through the endoplasmic reticulum. It is speculated that D1 may interact with the Bip region of the endoplasmic reticulum. Research has revealed that ATP is consumed when FVIII is transported out of the cell. Because the ATP pool in the cell is limited, proteins that consume less ATP can be exported out of the cell in greater quantities, producing more secretory proteins. The affinity and hydrophobicity of the protein were analyzed, and the DLG sequence in human factor VIII was mutated to SLG (D20S) or SLL (D20S, G22L). Since these are human FVIII mutants, the amino acid positions are based on the sequence of SEQ ID NO:1. For example, G22L here refers to the mutation of the 22nd amino acid G of the mutant FVIII to L (see SEQ ID NO:1).

[0119] Mutations in the D1 domain of human factor VIII include SLL (D20S, G22L), SLG (D20S) or both in combination with other amino acid mutations, and the various FVIII mutants are shown in Table 1. TIFF2024544499000001.tif114170 Table 1 shows various FVIII mutants containing DLG sequence mutations. The DLG sequence in the human D1 region is mutated to SLL (D20S, G22L) or SLG (D20S), or combined with other amino acid mutations to form various FVIII mutants with 3 to 5 mutated amino acids.

[0120] To compare the clotting activity of the FVIII mutants and human factor VIII (hFVIII-SQ), aPTT experiments were performed. As shown in Figure 9, all human factor VIII mutants had higher clotting activity than hFVIII-SQ. These results suggest that various combinations of these mutated amino acids increase the secretion of human factor VIII.

[0121] As described above, human factor VIII has 10 mutations, namely D20S, G22L, L50V, I61T, D115E, F129I, G132D, Q139E, L152P, and L159F, and these sites were determined to be important mutation sites that enhance the secretion ability and activity of human factor VIII.

[0122] Example 5: Production of recombinant AAV (rAAV) vectors containing engineered hFVIII polypeptides disclosed herein 0.8 x 10 in a 3 L bioreactor 6293 suspension cells were inoculated at a density of 1000 cells / mL. The three plasmids (pAAV-hFVIII, pAd-helper, and pRep / Cap) were mixed in a 1:1:1 ratio and then mixed with PEI in a 1:2 ratio (1ug plasmid:2μL PEI). The mixture was incubated at room temperature for 15 minutes and then added to the bioreactor. At 72 hours post-transfection, the cells were lysed with lysis buffer containing 1% Tween-20. The bioreactor was then added with 50U / mL Benzonase nuclease and 1mM MgCl2 and incubated at 37°C for 3 hours to digest DNA and RNA from unpackaged cells, viruses, and plasmids. The digested cell lysate was centrifuged and filtered to concentrate, after which the rAAV vector was purified on an AAVX affinity column and further purified on an anion column. After buffer exchange, the rAAV was sterile filtered and stored at -80°C.

[0123] Example 6: Preparation of pharmaceutical compositions containing engineered hFVIII polypeptides disclosed herein The rAAV vector purified in Example 5 is mixed with other active agents, preservatives, buffers, salts, pharma- ceutically acceptable carriers, or other pharma- ceutically acceptable ingredients to produce a pharmaceutical composition for treating patients with hemophilia A.

[0124] Example 7: Treatment of patients with hemophilia A with pharmaceutical compositions comprising engineered hFVIII polypeptides as disclosed herein Patients with hemophilia A were recruited to conduct a phase 1 clinical trial. The dose of the engineered hFVIII polypeptide to be tested was 0.5 × 10 12 Vg / Kg~6×10 13 Vg / Kg.

[0125] A pharmaceutical composition comprising a rAAV vector expressing a genetically engineered hFVIII polypeptide as disclosed herein was administered intravenously to a patient with hemophilia A. The patient was injected with the drug and then followed up for 4 years to track and detect the expression level of FVIII. The first checkpoint was on day 7 after the first injection, followed by detection every 1-2 weeks for 6 months, with subsequent intervals of 1-3 months. One year after the first injection, the patient's FVIII expression was above 2% of normal levels.

Claims

1. A genetically engineered human factor VIII (hFVIII) polypeptide comprising at least two substituted amino acids in the A1 domain of human factor VIII (hFVIII).

2. 2. The engineered hFVIII polypeptide of claim 1, wherein the substituted amino acids include L50 and L152 in the A1 domain.

3. 3. The genetically engineered hFVIII polypeptide of claim 1 or 2, wherein the substituted amino acids include L50V and L152P in the A1 domain.

4. 4. The genetically engineered hFVIII polypeptide of claim 2 or 3, wherein the substituted amino acids further comprise one or more of the amino acid substitutions selected from the group consisting of D20, G22, I61, D115, F129, G132, Q139 and L159 in the A1 domain.

5. 5. The genetically engineered hFVIII polypeptide of claim 4, wherein the amino acid substitutions at D20, G22, I61, D115, F129, G132, Q139 and L159 are D20S, G22L, I61T, D115E, F129I, G132D, Q139E and L159F, respectively.

6. The genetically engineered hFVIII polypeptide of any one of claims 1 to 5, wherein the substituted amino acids include D20S, L50V and L152P.

7. The genetically engineered hFVIII polypeptide of any one of claims 1 to 6, wherein the substituted amino acids include D20S, G22L, L50V and L152P.

8. The engineered hFVIII polypeptide of any one of claims 1 to 5, comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:

6.

9. An isolated nucleic acid fragment encoding the engineered hFVIII polypeptide of any one of claims 1-8.

10. 10. An expression vector comprising the nucleic acid fragment of claim 9 operably linked to a promoter.

11. 10. A recombinant AAV (rAAV) vector comprising the nucleic acid fragment of claim 9 operably linked to a promoter.

12. A pharmaceutical composition comprising an expression vector according to claim 10 or an rAAV vector according to claim 11.

13. A medicament for treating a patient with hemophilia A comprising the pharmaceutical composition of claim 12.

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