Recombinant adeno-associated virus vector, product and application of recombinant adeno-associated virus vector in treatment of hemophilia A

Abstract

The invention provides a recombinant adeno-associated virus vector, a product and application of the recombinant adeno-associated virus vector to treatment of hemophilia A, and belongs to the technical field of biological medicine. A recombinant AAV vector is prepared on the basis of an hFVIII nucleotide sequence with B structural domain deletion as shown in SEQ ID NO.1 or SEQ ID NO.2 or an optimized nucleotide variant sequence, the recombinant AAV vector has the characteristic of targeting liver, a human VIII factor gene can be delivered to liver cells of a patient diagnosed as type A hemophilia by using a replication-deficient adeno-associated virus, and the human VIII factor gene is promoted to be efficiently expressed, so that the recombinant AAV vector can be used for treating the type A hemophilia. The use dosage is not higher than 2 * 10 < 13 > vg / kg, and the risk of liver injury caused by high dosage is avoided from the source. The hemorrhage symptom of hemophilia patients can be relieved, a brand new technical normal form is provided for the A-type hemophilia from symptomatic blood transfusion to safer and more effective gene therapy, and the development of precise treatment of hereditary blood coagulation disorder diseases is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a recombinant adeno-associated virus vector, a product and application in treatment of hemophilia A. BACKGROUND

[0002] Hemophilia A (HA) is the most common inherited bleeding disorder, which is X-linked recessive inheritance, and 1 in 5000 males worldwide suffers from hemophilia A, the incidence is four times that of hemophilia B, and more than half of the hemophilia A patients belong to severe hemophilia. HA is caused by factor VIII (hFVIII) deficiency, which is very suitable for gene replacement method, and even moderate increase of hFVIII level (> 1% of normal value) can improve the severe bleeding phenotype.

[0003] The hFVIII gene is located at the end of the long arm of the X chromosome Xq28, which encodes a peptide chain arranged in the form of A1-A2-B-A3-C1-C2. The main mutation types closely related to severe hemophilia patients include large fragment deletion, nonsense mutation, inversion of intron 22, followed by small fragment deletion and insertion, missense mutation, etc.

[0004] Reduced hFVIII factor activity can inhibit the positive feedback loop in the coagulation cascade, resulting in incomplete coagulation process, which is manifested as prolonged clotting time, large ecchymosis, spontaneous oral and nasal bleeding, joint stiffness and chronic pain, and severe internal bleeding and anemia.

[0005] At present, the treatment of this disease mainly is exogenous protein replacement therapy, the first choice is gene recombinant hFVIII preparation or virus inactivated blood-derived hFVIII factor preparation, when there is no above condition, cold precipitate or fresh frozen plasma can be selected.

[0006] Because the half-life of hFVIII factor in vivo is relatively short, the conventional prophylactic replacement therapy needs to be administered every two days or every three days, which makes the individual in life to maintain compliance has a huge burden. Although the third generation of "long-acting" functional active drugs can reduce the administration frequency, the prophylactic replacement therapy using these drugs still needs to be administered permanently every month, or more frequently every week. Therefore, it is particularly important to achieve long-term expression of sustained, therapeutic level, endogenous hFVIII protein in treatment, so that patients no longer need such frequent exogenous protein input treatment. And continuous endogenous hFVIII protein expression can prevent bleeding complications and may establish immune tolerance to the protein.

[0007] Adeno-associated virus (AAV) is a small, replication-defective, nonenveloped animal virus that infects humans and some other primates. Several features of the AAV virus make this virus one of the most promising viral vectors for delivering therapeutic proteins through gene therapy: 1) the AAV virus does not cause human disease, inducing only a mild immune response, 2) the AAV viral vector can infect both dividing cells and cells in the resting phase, 3) the AAV is mainly found in free form within the cell, essentially not integrating into the host cell genome. Adeno-associated viral vectors have been successfully used in the treatment of some genetic diseases in the field of gene therapy due to their good safety and ability to mediate long-term expression of transgenes in post-mitotic tissues such as the liver.

[0008] About 30-60% of individuals in the human population have AAV antibodies. It is estimated that the seroprevalence of AAV1 neutralizing antibodies in healthy populations is about 67%, AAV2 is 72%, AAV5 is 40%, AAV6 is 46%, AAV8 is 38%, and AAV9 is 47%. Based on the homology of the AAV capsid structure, antibodies against one serotype of AAV can cross-react with other AAVs.

[0009] Due to the unique molecular and biochemical properties of human factor VIII, there are significant challenges in using AAV vectors for hemophilia A gene therapy. Compared with other similar-sized proteins, the expression efficiency of wild-type hFVIII is very low. By bioengineering the hFVIII molecule, the expression efficiency can be significantly improved. Specifically, the B domain of hFVIII is not essential for cofactor activity, and when the B domain is replaced by a short 14-amino acid linker (FVIII SQ), the mRNA transcription level is increased by 17 times compared with full-length wild-type FVIII, and the protein secretion is increased by 30%. Recombinant hFVIII-DBB-SQ has been used as a substitute for FVIII factor in clinical use (Refacto, Wyeth Pharma; Xyntha, Pfizer). However, in the reported clinical treatment of packaging FVIII-DBB-SQ expression cassette into AAV5 vector, the conduction efficiency is low, and a higher therapeutic dose (higher than 5 x 1011 13 vg / kg dose) is required, which not only can cause liver damage, but also the long-term efficacy is not sustained.

[0010] Therefore, a safer and more effective recombinant AAV viral vector is needed for hemophilia A gene therapy. How to optimize the viral vector, enhance the transduction efficiency of the viral vector, and maintain an effective vector dose while reducing the host immune response is crucial for optimizing hemophilia A gene therapy. SUMMARY

[0011] The application aims to provide a recombinant adeno-associated virus vector, a product and an application in treating hemophilia A, the recombinant adeno-associated virus vector has the property of targeting liver, human VIII factor gene can be delivered into hepatocytes of patients diagnosed as hemophilia A by using a replication-defective adeno-associated virus, and the human VIII factor gene is promoted to be expressed efficiently.

[0012] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0013] The application provides a recombinant adeno-associated virus vector, the recombinant adeno-associated virus vector comprises the following structure.

[0014] The hFVIII nucleic acid sequence with B domain deletion shown in SEQ ID NO. 1;

[0015] or the nucleotide variant sequence of the hFVIII with B domain deletion and preferred codon optimization shown in SEQ ID NO. 2;

[0016] or a sequence with 85% and above homology with the hFVIII nucleic acid sequence with B domain deletion shown in SEQ ID NO. 1 and the same effect;

[0017] or a sequence with 85% and above homology with the sequence shown in SEQ ID NO. 2 and the same effect.

[0018] Further, the recombinant adeno-associated virus vector further comprises a promoter sequence shown in SEQ ID NO. 3 or a sequence with 85% and above homology with the promoter sequence shown in SEQ ID NO. 3 and the same effect.

[0019] Further, the recombinant adeno-associated virus vector further comprises a recombinant adeno-associated virus vector skeleton shown in SEQ ID NO. 4.

[0020] Further, the 5' end ITR of the recombinant adeno-associated virus vector skeleton shown in SEQ ID NO. 4 is shown in SEQ ID NO. 5, the 3' end ITR is shown in SEQ ID NO. 6, the length of the 5' end ITR is 119 bp, the length of the 3' end ITR is 130 bp, and both are modified from the ITRs of the wild type AAV2, which are optimized terminal inverted repeat sequences with low CpG content.

[0021] Further, the recombinant adeno-associated virus vector further comprises a polyadenylation signal shown in SEQ ID NO. 9.

[0022] Further, the recombinant adeno-associated virus vector can express a hFVIII-BDD protein, and the sequence of the hFVIII-BDD protein is shown in SEQ ID NO. 10.

[0023] The present application also provides a product comprising the recombinant adeno-associated viral vector described above.

[0024] The present application also provides use of the product comprising the recombinant adeno-associated viral vector described above in the preparation of a medicament and / or reagent for treating hemophilia A.

[0025] The recombinant adeno-associated viral vector (recombinant AAV vector) for delivering the hFVIII gene in the present application should have a targeting property to the liver, and the hFVIII transgene should be controlled by a liver-specific expression control element.

[0026] In one embodiment, the expression control element comprises one or more of the following: a hybrid human liver-specific promoter (HLP); a thyroxinetransporth enhancer (enTTR); a transthyretin (TTR) promoter; an alpha 1 -antitrypsin (alAT) promoter; and a polyadenylation signal. In another embodiment, the expression control element comprises one or more of the following: a hybrid human liver-specific promoter HLP and a polyadenylation signal. Such elements are further described herein.

[0027] In one embodiment, the hFVIII gene encodes a Factor VIII B domain deletion (BDD) form, in which the B domain is replaced by a short amino acid linker (SQ), referred to herein as hFVIII-BDD. In one embodiment, the hFVIII-BDD protein sequence is as set forth in SEQ ID NO. 10. In one embodiment, the coding sequence of cohFVIII-BDD is codon-optimized for human expression, which sequence can share less than 80% identity with the native hFVIII coding sequence, the sequence being as set forth in SEQ ID NO. 2.

[0028] In one embodiment of the present application, the AAV vector comprising the hFVIII or hFVIII-BDD nucleic acid or variant can be administered to a patient by infusion in a biocompatible carrier, for example, by intravenous injection. The AAV vector can optionally be encapsulated in a liposome or mixed with other phospholipids or micelles to increase the stability of the molecule.

[0029] In one embodiment, the AAV supplying the capsid is the liver-tropic serotype AAV8, the coding sequence being as set forth in SEQ ID NO. 11.

[0030] In one embodiment, the AAV supplying the capsid is the liver-tropic serotype AAV5, the coding sequence being as set forth in SEQ ID NO. 12. In another embodiment, the AAV supplying the capsid protein is other liver-tropic serotype AAVs.

[0031] In one embodiment, serotype means that the serum-specific neutralization activity has been tested against all existing and characterized serotypes and no antibodies were found that neutralize the target virus. As more naturally occurring viral isolates are discovered and / or capsid mutants are generated, serological differences can or can not exist with any of the currently existing serotypes. In another embodiment, the serological testing of neutralization activity is performed on mutant viruses with capsid sequence modifications to determine if they belong to another serotype according to the traditional definition of serotype.

[0032] In one embodiment, where a recombinant plasmid is used to construct or manufacture the recombinant vector, the vector genome does not include plasmid portions that do not correspond to the vector genome sequence of the recombinant plasmid. The non-vector genome portion of the recombinant plasmid is referred to as the plasmid backbone, which is important for cloning and amplification of the plasmid, a process that is necessary for replication and recombinant virus production, but it is not itself packaged or encapsulated into the viral particle. Thus, the vector genome refers to the nucleic acid that is packaged or encapsulated by the virus.

[0033] In one embodiment, the selected regulatory sequences result in a total AAV vector genome size of about 4.8 to about 5.5 kilobases. In another embodiment, the selected regulatory sequences result in a total AAV vector genome size of about 5.0 kilobases.

[0034] ITRs are genetic elements responsible for genome replication and packaging during vector production, are the only viral cis elements required for production of recombinant AAV viruses, and can or can not have the same AAV origin as the capsid. In one embodiment, ITR sequences from AAV2 or partial nucleotide deletion versions thereof are used. In one embodiment a shortened version of the 5’ ITR, where the C-loop loop and terminal A sequence are partially deleted. In another embodiment a shortened version of the 3’ ITR, where the terminal A’ sequence is partially deleted. In other embodiments, full length AAV 5’ and 3’ ITRs are used. In one embodiment, the 5’ ITR sequence is set forth in SEQ ID NO. 5. In one embodiment, the 3’ ITR sequence is set forth in SEQ ID NO. 6, with a sequencing map shown in Figure 2 .

[0035] In addition to the promoter, the expression cassette and / or vector can comprise other appropriate transcription initiation, termination, enhancer sequences, and efficient RNA processing signals. These sequences include splice and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., of an ATG initiation codon); sequences that enhance protein stability; and, where appropriate, sequences that enhance secretion of the encoded product. In one embodiment, a polyadenylation signal is included to mediate termination of the cohFVIII-BDD mRNA transcript, the sequence of which is set forth in SEQ ID NO. 9.

[0036] In another aspect, the present application provides an aqueous suspension suitable for use in a hemophilia A patient, comprising the AAV gene therapy vector described herein. In certain embodiments, the suspension comprises an aqueous suspension and about 1 x 10 12 to about 1 x 10 14 genome copies of the gene therapy vector per milliliter. In one embodiment, the suspension is suitable for intravenous injection. In another embodiment, the suspension further comprises a surfactant, a preservative, and / or a buffer dissolved in the aqueous suspension.

[0037] In another embodiment, provided herein is a method of treating a hemophilia A patient using the AAV gene therapy vector described herein. In one embodiment, about 5 x 10 12 to about 5 x 10 13 genome copies per kilogram (vg / kg) of patient body weight are delivered to the patient in the form of an aqueous suspension.

[0038] The beneficial effects of the present application compared to the prior art are:

[0039] (1) The present application is based on the preparation of a recombinant AAV vector using the hFVIII nucleic acid sequence with B-domain deletion shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an optimized nucleotide variant sequence, which has the property of targeting the liver, and can use a replication-defective adeno-associated virus to deliver the human VIII factor gene to the hepatocytes of patients diagnosed with hemophilia A, and promote high-efficiency expression of the human VIII factor gene, and the use of a dose of no more than 2 x 10 13 vg / kg can avoid the risk of liver damage caused by high doses from the source. It can also alleviate the bleeding symptoms of hemophilia patients, and provide a new technical paradigm for the transition of hemophilia A from "symptomatic blood transfusion" to a safer and more effective gene therapy, and promote the development of precision treatment for genetic coagulopathy diseases.

[0040] (2) The present application enables long-term correction of the bleeding defect in a clinically meaningful way. The target patient population is moderate to severe hemophilia A patients, and the expected vector dose is intended to achieve a factor VIII blood concentration of about 10-40% or 20%. The ultimate goal of the recombinant AAV-mediated therapy is to achieve a constant level of factor VIII activity in the blood, avoiding the trough levels that occur with the current use of exogenous factors, allowing the patient to be free of the prophylactic treatment regimen, by only requiring one administration, which can reduce the need for frequent intravenous administration for a long period of time, possibly ten years or more. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor based on these drawings.

[0042] Figure 1 Structure diagram of the vectors pAAV-HLP-wthFVIII-BDD and pAAV-HLP-cohFVIII-BDD in the embodiments of the present application;

[0043] Figure 2 Sequencing diagram showing ITRs in the present application;

[0044] Figure 3 Activity level of factor VIII after intravenous injection of the vector in the present application;

[0045] Figure 4 Bleeding amount study data at the end point at 28 weeks after intravenous injection of the vector in Test Example 1 of the present application.

[0046] Figure 5 Full-length vector genome concentration in the liver of the mouse at 28 weeks after intravenous injection of the vector in Test Example 2 of the present application. DETAILED DESCRIPTION

[0047] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, features and embodiments of the present application.

[0048] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, solvent amounts, and the like, there can be multiple minimum and maximum values for the range. Each minimum and maximum value is specifically and individually disclosed. The scope of the present application is not intended to be bound by the values of the ranges.

[0049] Unless defined otherwise, 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 application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict, the present specification will control.

[0050] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative only.

[0051] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0052] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotide, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA, and anti-sense DNA, spliced or unspliced mRNA, rRNA, tRNA, and inhibitory DNA or RNA (RNAi, such as small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or anti-sense RNA). Polynucleotides include naturally occurring, synthetic, and artificially modified or engineered polynucleotides (e.g., variant nucleic acids). Polynucleotides can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length. In discussing polynucleotides, it will be understood that the sequence and structure of a particular polynucleotide is described herein in the 5' to 3' direction.

[0053] The term "modification" or "variant" and grammatical variations thereof, as used herein, refers to a nucleic acid, polypeptide, or sequence thereof, derived from a reference sequence. Thus, a modified or variant sequence can have substantially the same, greater, or less expression level, activity, or function as compared to the reference sequence, but retains at least a portion of the activity or function of the reference sequence. A particular example of a modification or variant is a codon-optimized nucleic acid sequence encoding FVIII. A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that has been genetically altered as compared to the wild-type. The sequence can be genetically altered without changing the encoded protein sequence. Alternatively, the sequence can be genetically altered to encode a variant protein. A nucleic acid or polynucleotide variant can refer to a sequence that has been codon-modified to encode a protein, but retains at least a portion of the sequence identical to the reference sequence, e.g., the wild-type protein sequence, which has also been codon-modified to encode a variant protein. For example, some codons of the nucleic acid variant will be changed without changing the amino acids of the protein (FVIII) encoded thereby, and some codons of the nucleic acid variant will be changed to change the amino acids of the protein (FVIII) encoded thereby.

[0054] The term "vector" as used herein refers to a small carrier nucleic acid molecule, a plasmid, a virus (e.g., an AAV vector), or other vehicle capable of inserting or incorporating nucleic acid manipulation. Such vectors can be used for genetic manipulation (i.e., "cloning vectors") to introduce / transfer a polynucleotide into a cell and transcribe or translate the inserted polynucleotide in the cell. An "expression vector" is a particular vector that contains a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell. The vector nucleic acid sequence typically contains at least one origin of replication for propagation in a cell, and optionally additional components, such as a heterologous polynucleotide sequence, expression control elements (e.g., promoters, enhancers), introns, ITRs, selectable markers (e.g., antibiotic resistance), polyadenylation signals.

[0055] The viral vector is derived from or based on one or more nucleic acid elements comprising viral genes. Particular viral vectors include lentiviral, pseudotyped lentiviral, and parvoviral vectors, such as adeno-associated viral (AAV) vectors. Also provided are nucleic acid sequences comprising a sequence encoding a variant FVIII polypeptide.

[0056] The term "recombinant" as used herein as a modifier of a vector, e.g., a recombinant virus, e.g., a lentivirus or parvovirus (e.g., AAV) vector, and a sequence modifier, e.g., a recombinant polynucleotide or polypeptide, refers to compositions that have been manipulated (e.g., genetically engineered) in a manner that would not ordinarily occur in nature. A specific example of a recombinant vector, e.g., an AAV vector, refers to the insertion of a polynucleotide that would not normally be found in the wild-type viral (e.g., AAV) genome into the viral genome. An example of a recombinant polynucleotide is a nucleic acid (e.g., a gene) encoding a FVIII protein cloned into a vector, with or without 5', 3', and / or intronic regions, that would not normally be associated within the viral (e.g., AAV) genome. Although the term "recombinant" is not always used in reference to vectors, e.g., viruses and AAV vectors, and sequences, e.g., polynucleotides, regardless of the omission, the inclusion of recombinant forms of polynucleotides is expressly contemplated herein.

[0057] Recombinant vector (e.g., lentivirus, parvovirus, AAV) sequences can be packaged into what is referred to herein as "particles" for subsequent infection (transduction) of cells in vitro or in vivo. When recombinant vector sequences are encapsidated or packaged into AAV particles, the particles can also be referred to as "rAAV." Such particles include proteins that encapsidate or package the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, capsid proteins.

[0058] A recombinant viral "vector" or "AAV vector" is derived from a wild-type genome of a virus, e.g., AAV, by removing the wild-type genome from the virus (e.g., AAV) using molecular methods and then replacing it with a non-native nucleic acid, e.g., a nucleic acid variant sequence encoding FVIII. Typically for AAV, one or both of the terminal inverted repeat (ITR) sequences of the AAV genome are retained in the AAV vector. A "recombinant" viral vector (e.g., AAV) is distinguished from a viral (e.g., AAV) genome because all or a portion of the viral genome associated with the viral (e.g., AAV) genome nucleic acid is replaced with a non-native sequence, e.g., a nucleic acid variant sequence encoding FVIII. The incorporation of the non-native sequence thus defines the viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be referred to as a "rAAV vector."

[0059] A vector "genome" refers to the portion of a recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., AAV) particle. In the case where a recombinant plasmid is used to construct or manufacture a recombinant vector, the vector genome does not include portions of the plasmid that do not correspond to the vector genome sequence of the recombinant plasmid. Such non-vector genome portions of the recombinant plasmid refer to the plasmid backbone, which is important for cloning and amplification of the plasmid, which is a necessary process for propagation and recombinant viral production, but is not important for its own packaging or encapsidation into a viral (e.g., AAV) particle. Thus, a vector "genome" refers to the nucleic acid that is packaged or encapsidated by a virus (e.g., AAV).

[0060] In cells having a transgene, the transgene has been introduced / transformed by way of "transduction" or "transfection" of the cell by a vector, such as an AAV. The terms "transduction" and "transfection" refer to the introduction of a molecule, such as a nucleic acid, into a cell or host organism. The transgene can or can not integrate into the genomic nucleic acid of the recipient cell. If the introduced nucleic acid integrates into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid can exist extrachromosomally in the recipient cell or host organism, or only transiently.

[0061] Transduced cells refer to cells that have had a transgene introduced. Thus, a transduced cell (e.g., in a mammal, such as a cell or tissue or organ cell) refers to a genetic change in a cell after incorporation of an exogenous molecule, such as a nucleic acid (e.g., a transgene) into the cell. Thus, a "transduced" cell is one into which an exogenous nucleic acid has been introduced, or progeny thereof. The cell can be propagated and express the introduced protein, or transcribed nucleic acid. For use and methods of gene therapy, the transduced cell can be present in a subject.

[0062] Expression control elements refer to nucleic acid sequences that affect expression of an operably linked nucleic acid. Control elements, including expression control elements described herein, such as promoters and enhancers, can include one or more expression control elements. Generally, these elements are included to facilitate proper transcription and proper translation of a heterologous polynucleotide (e.g., promoters, enhancers, splicing signals of introns, stop codons to maintain the correct reading frame of a gene to allow in-frame translation of mRNA, and the like). These elements generally act in cis, referred to as "cis-acting" elements, but can also act in trans.

[0063] Expression control can be at the level of transcription, translation, splicing, message stability, and the like. Generally, expression control elements that regulate transcription are placed near the 5' end (i.e., "upstream") of a transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of a transcribed sequence or within a transcript (e.g., in an intron). Expression control elements can be located near a transcribed sequence or at a distance (e.g., 1-10, 10-25, 25-50, 50-100, 100 to 500 or more nucleotides) from a transcribed sequence, or even at a considerable distance. However, due to the length limitations of certain vectors, such as AAV vectors, expression control elements are generally within 1 to 1000 nucleotides from a transcribed nucleic acid.

[0064] Expression constructs can include regulatory elements for driving expression in particular cell or tissue types. Expression control elements, such as promoters, include elements that are active in particular tissues or cell types, referred to herein as "tissue-specific expression control elements / promoters." Tissue-specific expression control elements are typically active in a particular cell or tissue, e.g., liver. Expression control elements are typically active in a particular cell, tissue, or organ because they are recognized by transcriptional activator proteins or other transcriptional regulators that are unique to the particular cell, tissue, or organ type.

[0065] Other elements include, for example, stuffer or stuffer polynucleotide sequences, e.g., for improving packaging and reducing the presence of contaminating nucleic acids. AAV vectors typically accept DNA inserts that range in size typically from about 4.3 kilobases to about 5.3 kilobases, or slightly larger. Thus, for shorter sequences, a stuffer or filler should be included in order to adjust the length to be close to or reach the normal size of viral genome sequence that the AAV vector can accept for packaging into a viral particle. In various embodiments, the stuffer / filler nucleic acid sequence is an untranslated (non-protein coding) segment of nucleic acid. For nucleic acid sequences that are less than 4.9 kilobases, the stuffer or stuffer polynucleotide sequence has a length, when combined with the sequence (e.g., inserted into a vector), that is between about 3.5-5.5 kilobases in total length, or between about 4.5-5.3 kilobases, or between about 4.8-5.5 kilobases.

[0066] To further illustrate the technical means adopted by the present application and its effects, the present application is further described below in conjunction with the embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

[0067] The specific techniques or conditions not specified in the examples are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments used are not specified by the manufacturer, and can be commercially available through regular channels.

[0068] Example 1

[0069] Example 1 of the present application provides a method for preparing a recombinant vector pAAV-HLP-wthFVIII-BDD, and the specific steps are as follows:

[0070] (1) The nucleotide sequences of CAG promoter, dTomatoF gene, and IRER / W3SL element on the M6771A (GenBank #PP719195) vector plasmid were excised using restriction endonuclease Mlul and Notl, recovered, and a recombinant adeno-associated virus vector skeleton represented by the sequence of SEQ ID NO. 4 was obtained.

[0071] (2) Preparation of recombinant vector pAAV-HLP-wthFVIII-BDD:

[0072] 1) The hFVIII nucleic acid sequence with B domain deletion shown in SEQ ID NO. 1 (wthFVIII-BDD) was connected to the promoter sequence shown in SEQ ID NO. 3 by fusion PCR to obtain the sequence HLP-wthFVIII-BDD shown in SEQ ID NO. 7:

[0073] A, the upstream primer sequence SEQ ID NO. 17 and the downstream primer sequence SEQ ID NO. 18 were designed using the sequence SEQ ID NO. 1 as a template. The sequence SEQ ID NO. 1, the sequence SEQ ID NO. 17, and the sequence SEQ ID NO. 18 were used for PCR reaction, and the PCR reaction conditions were as follows: 95°C for 60s, 58°C for 70s, 72°C for 200s, for a total of 30 cycles, and the last 72°C extension for 10 min. The product was identified by electrophoresis on a 0.8% agarose gel, and the band fragment with a molecular weight of about 4300bp was recovered using the Qiagen gel recovery kit, and the operation was performed according to the manufacturer's instructions.

[0074] B, the upstream primer sequence SEQ ID NO. 19 and the downstream primer sequence SEQ ID NO. 20 were designed using the sequence SEQ ID NO. 3 as a template. The sequence SEQ ID NO. 3, the sequence SEQ ID NO. 18, and the sequence SEQ ID NO. 19 were used for PCR reaction, and the PCR reaction conditions were as follows: 95°C for 60s, 58°C for 70s, 72°C for 30s, for a total of 30 cycles, and the last 72°C extension for 10 min. The product was identified by electrophoresis on a 2% agarose gel, and the band fragment with a molecular weight of about 270bp was recovered using the Qiagen gel recovery kit, and the operation was performed according to the manufacturer's instructions.

[0075] C, the two gel-recovered DNA fragments of steps A and B were used as templates, and the upstream primer SEQ ID NO. 19 and the downstream primer SEQ ID NO. 18 were used for PCR reaction; the PCR reaction conditions were as follows: 95°C for 60s, 55°C for 70s, 72°C for 300s, for a total of 30 cycles, and the last 72°C extension for 10 min. The product was identified by electrophoresis on a 0.8% agarose gel, and the band fragment with a molecular weight of about 4700bp was recovered using the Qiagen gel recovery kit to obtain the sequence shown in SEQ ID NO. 7 (HLP-wthFVIII-BDD), and the operation was performed according to the manufacturer's instructions.

[0076] 2) The sequence shown as SEQ ID NO. 9 and the sequence shown as SEQ ID NO. 7 are connected by high-fidelity PCR to obtain the sequence shown as SEQ ID NO. 13:

[0077] According to the sequence shown as SEQ ID NO. 13, an upstream primer SEQ ID NO. 21 (containing an MluI site) and a downstream primer SEQ ID NO. 22 (containing a NotI site) are designed, and a PCR reaction is performed with the fragment of SEQ ID NO. 7 as a template, and the PCR reaction conditions are: 95℃ for 60s, 58℃ for 70s, 72℃ for 300s, a total of 32 cycles, and a final 72℃ extension for 10min. After the PCR is performed according to the above conditions, the product is identified by electrophoresis on a 0.8% agarose gel, and the band fragment with a molecular weight of about 5000bp is recovered by a Qiagen gel recovery kit, to obtain a fragment with the sequence shown as SEQ ID NO. 13. The operation is performed according to the manufacturer's instructions.

[0078] 3) The sequence shown as SEQ ID NO. 13 is double digested with MluI and NotI, and then connected with the vector backbone shown as SEQ ID NO. 4, to obtain a recombinant vector pAAV-HLP-wthFVIII-BDD shown as SEQ ID NO. 15. The group of vectors pAAV-HLP-wthFVIII-BDD is shown as SEQ ID NO. 15. Figure 1

[0079] Example 2

[0080] The embodiment 2 of the present application provides a preparation method of a recombinant vector pAAV-HLP-cohFVIII-BDD, and the specific steps are as follows:

[0081] (1) The sequence shown as SEQ ID NO. 2 and the sequence shown as SEQ ID NO. 3 are connected by fusion PCR to obtain the sequence shown as SEQ ID NO. 8:

[0082] 1) The sequence shown as SEQ ID NO. 2 is used as a template, an upstream primer sequence SEQ ID NO. 23 and a downstream primer sequence SEQ ID NO. 24 are designed, and a PCR reaction is performed, and the PCR reaction conditions are: 95℃ for 60s, 58℃ for 70s, 72℃ for 200s, a total of 30 cycles, and a final 72℃ extension for 10min. The product is identified by electrophoresis on a 0.8% agarose gel, and the band fragment with a molecular weight of about 4300bp is recovered by a Qiagen gel recovery kit, and the operation is performed according to the manufacturer's instructions.

[0083] ​2) The sequence of SEQ ID NO. 3 was used as a template to design the sequence of the upstream primer SEQ ID NO. 19 and the sequence of the downstream primer SEQ ID NO. 25 for PCR reaction. The PCR reaction conditions were as follows: 95 °C for 60 s, 58 °C for 70 s, 72 °C for 30 s, for a total of 30 cycles, and a final extension at 72 °C for 10 min. The product was identified by electrophoresis on a 2% agarose gel, and the band fragment with a molecular weight of about 270 bp was recovered using a Qiagen gel recovery kit, following the manufacturer's instructions.

[0084] 3) The DNA fragments recovered from the two steps in 1) and 2) were used as templates, and the sequence of the upstream primer SEQ ID NO. 19 and the sequence of the downstream primer SEQ ID NO. 24 were used for PCR reaction. The PCR reaction conditions were as follows: 95 °C for 60 s, 55 °C for 70 s, 72 °C for 300 s, for a total of 30 cycles, and a final extension at 72 °C for 10 min. After PCR was performed according to the above conditions, the product was identified by electrophoresis on a 0.8% agarose gel, and the band fragment with a molecular weight of about 4700 bp was recovered using a Qiagen gel recovery kit, to obtain a fragment with the sequence of SEQ ID NO. 8, following the manufacturer's instructions.

[0085] (2) The sequence of SEQ ID NO. 9 was combined with the sequence of SEQ ID NO. 8 by high-fidelity PCR to obtain the sequence of SEQ ID NO. 14:

[0086] The sequence of the upstream primer SEQ ID NO. 21 (containing an MluI site) and the sequence of the downstream primer SEQ ID NO. 26 (containing a NotI site) were designed according to the sequence of SEQ ID NO. 14, and PCR was performed using the fragment of SEQ ID NO. 8 as a template. The PCR reaction conditions were as follows: 95 °C for 60 s, 58 °C for 70 s, 72 °C for 300 s, for a total of 32 cycles, and a final extension at 72 °C for 10 min. After PCR was performed according to the above conditions, the product was identified by electrophoresis on a 0.8% agarose gel, and the band fragment with a molecular weight of about 5000 bp was recovered using a Qiagen gel recovery kit, to obtain a fragment with the sequence of SEQ ID NO. 14, following the manufacturer's instructions.

[0087] (3) The sequence of SEQ ID NO. 14 was digested with MluI and NotI and ligated to the vector backbone shown in the sequence of SEQ ID NO. 4 to obtain the recombinant vector pAAV-HLP-cohFVIII-BDD shown in the sequence of SEQ ID NO. 16. Figure 1

[0088] Example 3

[0089] ​Embodiment 3 of the present application provides a method for preparing a complete AAV vector containing the recombinant vector pAAV-HLP-wthFVIII-BDD, the specific steps are as follows:

[0090] By conventional methods, the AAV viral genome lacking ITRs, including the rep gene of AAV2 (SEQ ID NO. 27), and the cap gene of AAV8 (SEQ ID NO. 11) or the cap gene of AAV5 (SEQ ID NO. 12) are cloned into a plasmid to provide the rep and cap proteins required for AAV vector packaging, and the promoter (p5) of the rep gene is relocated in the plasmid to prevent the formation of replication-competent AAV by non-homologous recombination, thereby obtaining AAV packaging plasmids pAAV-RC8 / pRep2Cap8 (Plasmid #112864, HonorGene) and pAAV-RC5 / pRep2Cap5 (Plasmid #VPK-425, Cell Biolabs).

[0091] By conventional methods, the three genes of adenovirus, E2A (SEQ ID NO. 28), E4 (SEQ ID NO. 29) and VA (SEQ ID NO. 30), are cloned into a third plasmid pHelper to provide the necessary adenovirus helper functions for AAV vector replication, encapsidation and mature virion assembly in HEK293 cells, while avoiding the risk of contamination by wild-type adenovirus.

[0092] pAAV-HLP-wthFVIII-BDD, pAAV-RC8 / pRep2Cap8, pHelper are transfected into human embryonic kidney HEK293 cells to produce AAV vectors (AAV8-wtFVIII-BDD) by a virus-free process. pAAV-HLP-cohFVIII-BDD, pAAV-RC5 / pRep2Cap5, pHelper are transfected into human embryonic kidney HEK293 cells to produce AAV vectors (AAV5-wtFVIII-BDD) by a virus-free process. The HEK293 cell line is a permanent cell line obtained by transformation of primary human embryonic kidney with adenovirus type 5 (Ad5) DNA, and the HEK293 working cell bank is derived from the American Type Culture Collection (ATCC) cell bank.

[0093] Embodiment 4

[0094] Example 4 of the present application was prepared by the method of Example 1 to prepare the complete AAV vector containing the recombinant vector pAAV-HLP-cohFVIII-BDD, which is different from Example 1, that is, Example 4 is to transfect human embryonic kidney HEK293 cells with pAAV-HLP-cohFVIII-BDD to obtain complete AAV vector (AAV8-coFVIII-BDD and AAV5-coFVIII-BDD).

[0095] Test Example 1

[0096] The present application test example 1 detects the effect of the AAV vector prepared in Example 3, the specific steps are as follows:

[0097] The revision or procedure of animal care and use in this test example will be reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) before implementation. The HA mouse (B6 / JGpt-F8em430Cd5d452 / F8em430Cd5d452 / Gpt) is a homozygote of a targeted X chromosome-linked mutant allele. The FVIII activity of homozygous female is less than 0.1% of normal mice, which reproduces the bleeding phenotype of human hemophilia A.

[0098] (1) The homozygous hemophilia HA mouse and the wild type C57BL / 6J mouse were used as experimental animals, and the mice were placed in Makrolon cages with micro-isolation caps and contact bedding. The animals can freely obtain standard rodent food and drinking water. The animal room is maintained under artificial lighting for 12 hours of light / dark cycle. The ambient temperature is maintained at 23℃±2℃, and the humidity is maintained at 40%-70%.

[0099] (2) AAV8-coFVIII-BDD, AAV5-wtFVIII-BDD were prepared according to the method of Example 3, and AAV vector suspension was prepared with phosphate buffered saline / 0.001% Pluronic F-68 as buffer, and a single intravenous injection (4 μL / g body weight) was given to 6-8 week old (average body weight 20±2 g) female hemophilia HA mice through the tail vein. In order to compare the effect of gene therapy and preventive treatment, Xyntha was given to a group of hemophilia HA mice through the tail vein 5 minutes before the bleeding was measured, and the dose was 100 IU / kg.

[0100] Peripheral blood was collected from retro-orbital plexus every four weeks, and plasma was collected into sodium citrate collection tubes, and the plasma level of hFVIII was detected by human specific ELISA (human FVIII ELISA kit, #ab272771, Abeam).

[0101] The data of the study of the activity level of hVIII factor after intravenous injection of the vector in the mouse tail is as follows: Figure 3shown.

[0102] Figure 3 All animals in the study showed expression of hFVIII after AAV vector delivery, with mean hFVIII antigen levels peaking at approximately 8 weeks post vector administration.

[0103] At the end of week 28, each mouse was anesthetized by intraperitoneal injection of a ketamine-xylazine (KX) solution and maintained under anesthesia throughout the tail bleeding test, and CO2 euthanasia was performed after the end of the bleeding experiment. The body temperature of the mice was maintained by placing them on a temperature-controlled heating pad. To standardize local blood circulation, the tail of the mouse was immersed in 37 °C saline for 10 minutes. Subsequently, the tail was removed from the warm saline and cut off 5 mm from the tail tip with a disposable scalpel. Immediately after amputation, the tail was placed in a pre-weighed 15 mL conical tube containing 10 mL of 37 °C saline and immersed for 30 minutes. At the end of 30 minutes, the test tube was weighed again, and the amount of bleeding was calculated from the difference in weight before and after. Bleeding level study data are shown in Figure 4

[0104] Figure 4 It is shown that after injection of the AAV vector of the present application, the amount of bleeding in mice is significantly reduced, close to the bleeding level of wild-type mice.

[0105] Test Example 2

[0106] Test Example 2 of the present application detects vector genome morphology, the specific steps are as follows:

[0107] The number of vector genome morphologies is measured by ddPCR, by capturing single DNA molecules in thousands of water-oil emulsion droplets, followed by PCR-mediated amplification in each droplet, and discrete measurement of end-point fluorescence of individual droplets. Each droplet is counted as negative or positive by fluorescence, and the fraction of positive droplets is applied to Poisson statistics to estimate the copy number of target DNA molecules per sample.

[0108] ​The total volume of the PCR reaction system was 20 microliters, ddPCR Supermix for Probes (Bio-Rad Laboratories) without deoxyuridine triphosphate (dUTP) was used, the concentration of each primer (forward and reverse) was 450 nM, the concentration of the fluorescent probe was 250 nM, and 10 ng of genomic DNA was added. The reaction mixture was mixed with self-generated oil (Bio-Rad Laboratories), and droplets were prepared by Bio-Rad Auto Droplet Generator (Bio-Rad Laboratories) and then transferred to a 96-well plate. The PCR reaction was performed in a C1000 Touch thermal cycler, and the program was as follows: 95°C pre-denaturation for 10 minutes; 60 cycles, each cycle 95°C denaturation for 30 seconds, 58°C annealing and extension for 1 minute; then 98°C extension for 10 minutes, and finally kept at 4°C.

[0109] The endogenous housekeeping gene AP3B1 (i.e., the beta 1 subunit of the transporter complex 3) was detected as a standardization reference for calculating the copy number of the vector in each diploid genome.

[0110] The AP3B1 forward primer sequence was SEQ ID NO. 31, the reverse primer sequence was SEQ ID NO. 32, and the probe sequence was SEQ ID NO. 33 (5' end labeled with HEX, 3' end labeled with BGQ1).

[0111] The quantitative detection of the vector genome used specific primers and probes targeting the core region of the vector genome, the forward primer sequence was SEQ ID NO. 34, the reverse primer sequence was SEQ ID NO. 35, and the probe primer sequence was SEQ ID NO. 36 (5' end labeled with 6-FAM, 3' end labeled with BGQ1).

[0112] The quantitative detection of the codon-optimized vector genome used specific primers and probes targeting the core region of the vector genome, the forward primer sequence was SEQ ID NO. 37, the reverse primer sequence was SEQ ID NO. 38, and the probe primer sequence was SEQ ID NO. 39 (5' end labeled with 6-FAM, 3' end labeled with BGQ1).

[0113] To further determine the continuity and integrity of the vector genome, a 4-plex ddPCR assay was performed. In addition to detecting the internal control housekeeping gene AP3B1 and the core region target, amplicons to the 5' and 3' ends of the vector genome were introduced using custom specific primers / probes. The vector genome 5' end amplicon forward primer sequence was SEQ ID NO. 40, reverse primer sequence was SEQ ID NO. 41, and probe primer sequence was SEQ ID NO. 42 (5' end labeled CY5, 3' end labeled BGQ2). The vector genome 3' end amplicon forward primer sequence was SEQ ID NO. 43, reverse primer sequence was SEQ ID NO. 44, and probe primer sequence was SEQ ID NO. 45 (5' end labeled ROX, 3' end labeled BGQ2).

[0114] To determine the continuity and integrity of the codon-optimized vector genome, a 4-plex ddPCR assay was performed with the vector genome 5' end amplicon forward primer sequence of SEQ ID NO. 40, reverse primer sequence of SEQ ID NO. 41, and probe primer sequence of SEQ ID NO. 42. The vector genome 3' end amplicon forward primer sequence was SEQ ID NO. 46, reverse primer sequence was SEQ ID NO. 44, and probe primer sequence was SEQ ID NO. 47 (5' end labeled ROX, 3' end labeled BGQ2).

[0115] The 4-plex ddPCR reaction samples were read by the QX600 Droplet Reader (Bio-Rad Laboratories), and the total concentration of target sequences and the number of linked copies of target sequences were processed by QuantaSoft software vl.7.4.0917 (Bio-Rad Laboratories) and analyzed in conjunction with the more advanced higher-order linkage calibration algorithm (Bio-Rad Laboratories). The concentration (copy number / cell) of the full-length vector genome in the mouse liver 28 weeks after intravenous injection of the vector in the mouse tail vein was determined as shown in Table 2, and the detection method was based on two-probe linkage analysis of the integrity of the vector genome and three-probe linkage analysis of the integrity of the vector genome. Figure 5

[0116] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A recombinant adeno-associated viral vector, characterized in that, The recombinant adeno-associated virus vector comprises the following structure: a hFVIII nucleic acid sequence with B domain deletion as shown in SEQ ID NO. 1; or a nucleotide variant sequence of hFVIII with B domain deletion and preferred codon optimization as shown in SEQ ID NO. 2; or a sequence with 85% homology and above to the hFVIII nucleic acid sequence with B domain deletion as shown in SEQ ID NO. 1 and the same effect; or a sequence with 85% homology and above to the sequence as shown in SEQ ID NO. 2 and the same effect.

2. The recombinant adeno-associated viral vector of claim 1, wherein, The recombinant adeno-associated virus vector further comprises a promoter sequence as shown in SEQ ID NO. 3 or a sequence with 85% homology and above to the promoter sequence as shown in SEQ ID NO. 3 and the same effect.

3. The recombinant adeno-associated viral vector of claim 1, wherein, The recombinant adeno-associated virus vector further comprises a recombinant adeno-associated virus vector skeleton as shown in SEQ ID NO.

4.

4. The recombinant adeno-associated viral vector of claim 3, wherein, The 5' ITR of the recombinant adeno-associated virus vector skeleton as shown in SEQ ID NO. 4 is as shown in SEQ ID NO. 5, and the 3' ITR is as shown in SEQ ID NO. 6, the 5' ITR is 119 bp in length, and the 3' ITR is 130 bp in length, both of which are modified from the ITRs of wild-type AAV2 and are optimized, low-CpG-content inverted terminal repeats.

5. The recombinant adeno-associated viral vector of claim 1, wherein, The recombinant adeno-associated virus vector further comprises a polyadenylation signal as shown in SEQ ID NO.

9.

6. The recombinant adeno-associated viral vector of claim 1, wherein, The recombinant adeno-associated virus vector can express a hFVIII-BDD protein, and the sequence of the hFVIII-BDD protein is as shown in SEQ ID NO.

10.

7. A product comprising the recombinant adeno-associated virus vector of any one of claims 1-6.

8. Use of the product comprising the recombinant adeno-associated virus vector of claim 7 in the preparation of a medicament and / or reagent for treating hemophilia A.

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