Adeno-associated virus factor viii vectors

Abstract

To provide novel AAV vector encoding functional factor VIII protein useful in gene therapy for treating hemophilia A.SOLUTION: The present invention provides AAV vectors (referred to herein as "AAV FVIII vectors") that encode functionally active FVIII. The genome encoding the functionally active FVIII is preferably 7.0 kb in length or less, more preferably 6.5 kb in length or less, even more preferably 6.0 kb in length or less, even more preferably 5.5 kb in length or less, and even more preferably 5.0 kb in length or less, and has enhanced promoter function. The present invention also relates to methods for making the AAV FVIII vector described herein and related therapeutic uses.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 61 / 877,042, filed on September 12, 2013, which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to adeno-associated virus (AAV) factor VIII (FVIII) vectors, including AAV FVIII vectors having high expression activity and AAV FVIII vectors expressing full-length or truncated functional FVIII. The present invention also relates to methods of making the AAV FVIII vectors described herein and their related therapeutic uses.

Background Art

[0003] Adeno-associated virus (AAV) is a small, replication-defective, non-enveloped animal virus that infects humans and several other primate species. Due to several characteristics of AAV, this virus has become an attractive vehicle for the delivery of therapeutic proteins by gene therapy. For example, AAV is not known to cause human disease, induces a mild immune response, and AAV vectors can infect both dividing and resting cells without integration into the host cell genome. Gene therapy vectors using AAV have been successfully used in several clinical trials, for example, for the delivery of human factor IX (FIX) to the liver for the treatment of hemophilia B (Nathwani et al., New Engl. J. Med. 365:2357-2365, 2011).

[0004] However, AAV gene therapy vectors have several drawbacks. Specifically, the cloning capacity of AAV vectors is limited by the DNA packaging capacity of the virus. The single-stranded DNA genome of wild-type AAV is approximately 4.7 kilobases (kb). In practice, an AAV genome of about 5.0 kb or less is thought to be completely packaged (i.e., full-length) within AAV virus particles. Due to the requirement that the nucleic acid genome within an AAV vector must have two AAV inverted terminal repeats (ITRs) of about 145 bases, the DNA packaging capacity of an AAV vector is such that a maximum of about 4.4 kb of protein-coding sequence can be capsid-formed.

[0005] Due to this size limitation, large therapeutic genes, i.e., genes longer than about 4.4 kb in length, are generally not suitable for use with AAV vectors. One such therapeutic gene is the Factor VIII (FVIII) gene, which has an mRNA of about 7.0 kb encoding a 2332-amino acid polypeptide that contains, from the N-terminus to the C-terminus, a 19-amino acid signal peptide and three large domains (i.e., the heavy chain or A domain, the central or B domain, and the light chain or C domain). One strategy used to overcome the size limitation of AAV vectors in FVIII was to use two AAV vectors, one AAV vector encoding the heavy chain or A domain and the other AAV vector encoding the light chain or C domain (see, for example, Coutu et al., U.S. Patent Nos. 6,221,349, 6,200,560, and 7, 351,577). Another strategy to circumvent this size limitation was to generate an AAV vector encoding FVIII in which the central portion or B domain of FVIII was deleted and replaced with a 14-amino acid linker known as the SQ sequence (Ward et al., Blood, 117:798-807, 2011, and McIntosh et al., Blood 121:3335-3344, 20 13).

[0006] AAV vectors having AAV genomes longer than 5.0 kb have been reported in the literature, but in many of these cases, the 5' or 3' ends of the encoded gene appear to be truncated (see Hirsch et al., Molec. Ther. 18-6-8, 2010, and Ghosh et al., Bi otech. Genet. Engin. Rev. 24:165-178, 2007). However, in AAV-infected cells , overlapping homologous recombination occurs between nucleic acids having 5' and 3' truncations, resulting in the production of a "complete" nucleic acid encoding the said large protein, thereby demonstrating that a functional full-length gene is reconstructed.

[0007] There is a need for novel AAV vectors encoding a functional Factor VIII protein that are useful in gene therapy for treating hemophilia A. Accordingly, the present invention provides an AAV virus particle that encapsidates an entire nucleic acid encoding a therapeutic protein (i.e., a fully packaged AAV FVIII vector), thereby avoiding the above problem of genome size excess, or encoding a functionally active Factor VIII protein that may or may not be truncated, at least producing a functionally active FVIII. Further, in order to avoid an immune response against the capsid, the AAV vector should have the highest target protein transduction / expression activity per capsid particle. The present invention also relates to the production of a complete AAV FVIII vector having high expression activity. Finally, the present invention relates to a method for producing the AAV Factor VIII vector described herein, and related methods for using the same.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0009] The present invention provides an AAV vector encoding functionally active FVIII (referred to herein as “AAV FVIII vector”). The genome encoding functionally active FVIII is preferably 7.0 kb or less in length, more preferably 6.5 kb or less in length, even more preferably 6.0 kb or less in length, even more preferably 5.5 kb or less in length, even more preferably 5.0 kb or less in length, and has enhanced promoter function.

[0010] As used herein, “functionally active FVIII” is an FVIII protein that has the functionality of the wild-type FVIII protein when expressed in cultured cells in vitro or in vivo when expressed in cells or body tissues. This includes, for example, enabling blood clotting to occur and shortening the time it takes for blood to clot in a subject suffering from hemophilia A. Wild-type FVIII is involved in blood clotting through the coagulation cascade and acts as a cofactor for activated FIX (FIXa) to form a complex that converts factor X (FX) to activated FX (FXa) in the presence of calcium ions and phospholipids. Thus, functionally active FVIII can form a complex with FIXa, and this complex can convert FX to FXa.

[0011] As used herein, an "AAV vector" refers to a single-stranded or double-stranded nucleic acid having an AAV 5' inverted terminal repeat (ITR) sequence and an AAV 3' ITR adjacent to a transcriptional regulatory element, i.e., a protein coding sequence operably linked to one or more promoters and / or enhancers, and a polyadenylation sequence, and, optionally, one or more introns inserted between exons of the protein coding sequence. A single-stranded AAV vector refers to a nucleic acid that is present within the genome of an AAV viral particle and can be either the sense strand or the antisense strand of the nucleic acid sequences disclosed herein. The size of such a single-stranded nucleic acid is given in base units. A double-stranded AAV vector refers to a nucleic acid that is present within the DNA of a plasmid (e.g., pUC19) or within the genome of a double-stranded virus (e.g., baculovirus) that is used to express or transduce the AAV vector nucleic acid. The size of such a double-stranded nucleic acid is given in base pairs (bp).

[0012] The term "inverted terminal repeat (ITR)" as used herein refers to regions present at the 5' and 3' termini of the AAV genome that function cis as origins of DNA replication and as packaging signals for the viral genome and are recognized in the art. The AAV ITRs, together with the AAV rep coding region, efficiently provide for the removal and rescue from the host cell genome and the integration of nucleotide sequences inserted between two adjacent ITRs into the host cell genome. The sequences of certain AAV-related ITRs are disclosed by Yan et al., J. Virol. 79(1):364-379 (2005) (which is hereby incorporated by reference in its entirety). into this specification).

[0013] "Transcription regulatory element" refers to the nucleotide sequence of a gene involved in the regulation of gene transcription, and includes, for example, a promoter, a response element, an activator and an enhancer sequence, which assist the binding of RNA polymerase and promote expression by binding of a transcription factor, and an operator or silencer sequence, which blocks the binding of RNA polymerase and inhibits expression by binding of a repressor protein. The term "liver-specific transcription regulatory element" refers to a regulatory element that regulates gene expression specifically in liver tissue. Examples of liver-specific regulatory elements include, but are not limited to, the mouse thyretin promoter (mTTR), the endogenous human factor VIII promoter (F8), the human α1-antitrypsin promoter (hAAT) and its active fragments, the human albumin minimal promoter, and the mouse albumin promoter. Together with Enh1, enhancers derived from liver-specific transcription factor binding sites such as EBP, DBP, HNF1, HNF3, HNF4, HNF6, etc. are also considered.

[0014] In one embodiment, the AAV vector of the present invention comprises a nucleic acid encoding a functionally active FVIII having a B domain replaced by a 14 amino acid SQ sequence, i.e., encoding FVIII SQ. The SQ sequence is disclosed in Ward et al., Blood, 117:798-807, 2011, and McIntosh et al., Blood 121:3335-3344, 2013. The FVI II coding region sequence is a codon-optimized sequence (see Nathwani et al., US Patent Application Publication No. 2013 / 0024960A1, published on January 24, 2013, which is hereby incorporated by reference in its entirety, and McIntosh et al., Blood 121:3335-3344, 2013). This sequence is referred to herein as "UCL SQ FVIII".

[0015] In a first aspect, the AAV vector of the present invention comprises Proto1, schematically shown in FIG. 2A, and comprises the nucleic acid sequence set forth in SEQ ID NO: 1.

[0016] In a second aspect, the AAV vector of the present invention comprises Proto1S, schematically shown in FIG. 2B, and comprises the nucleic acid sequence set forth in SEQ ID NO: 2.

[0017] In a third aspect, the AAV vector of the present invention comprises Proto2S, schematically shown in FIG. 2C, and comprises the nucleic acid sequence set forth in SEQ ID NO: 3.

[0018] In a fourth aspect, the AAV vector of the present invention comprises Proto3S, schematically shown in FIG. 2D, and comprises the nucleic acid sequence set forth in SEQ ID NO: 4.

[0019] In another embodiment, the AAV vector of the present invention comprises a nucleic acid encoding FVIII that lacks the entire B domain containing the SQ sequence and the a3 domain located immediately N-terminal to the light chain or C domain. The FVIII coding region sequence is a codon-optimized sequence (see U.S. Patent Application Publication No. 2013 / 0024960A1, published January 24, 2013 (the entire disclosure of which is incorporated herein by reference), and McIntosh et al., Blood 121:3335-3344, 2013).

[0020] In a first aspect, the AAV vector of the present invention comprises Proto4, schematically shown in FIG. 3A, and comprises the nucleic acid sequence set forth in SEQ ID NO: 5.

[0021] In a second aspect, the AAV vector of the present invention comprises Proto5, schematically shown in FIG. 3B, and comprises the nucleic acid sequence set forth in SEQ ID NO: 6.

[0022] In a third aspect, the AAV vector of the present invention comprises Proto6, schematically shown in FIG. 3C, and comprises the nucleic acid sequence set forth in SEQ ID NO: 7.

[0023] In a fourth aspect, the AAV vector of the present invention comprises Proto7 schematically shown in FIG. 3D and comprises the nucleic acid sequence set forth in SEQ ID NO: 8.

[0024] In another embodiment, the AAV vector of the present invention comprises a nucleic acid comprising an AAV2 5' inverted terminal repeat (ITR), a liver-specific transcriptional regulatory region, a codon-optimized functionally active FVIII coding region, optionally one or more introns, a polyadenylation sequence, and an AAV2 3' ITR. In a preferred embodiment, the liver-specific transcriptional regulatory region comprises a truncated ApoE enhancer sequence, an 186-base human α1-antitrypsin (hAAT) proximal promoter (including 42 bases of the 5' untranslated region (UTR)), and one or more enhancers selected from the group consisting of (i) a 34-base human ApoE / C1 enhancer, (ii) a 32-base human AAT promoter distal X region, and (iii) 80 additional bases of the distal element of the human AAT proximal promoter; the codon-optimized functionally active FVIII coding region encodes an FVIII SQ variant. In another preferred embodiment, the liver-specific transcriptional regulatory region comprises an α1-microglobulin-enhancer sequence and an 186-base human α1-antitrypsin (AAT) proximal promoter.

[0025] In a first aspect, the AAV vector of the present invention comprises construct 100ATG comprising the nucleic acid sequence set forth in SEQ ID NO: 9.

[0026] In a second aspect, the AAV vector of the present invention comprises construct 100ATG bGH polyA comprising the nucleic acid sequence set forth in SEQ ID NO: 10.

[0027] In a third aspect, the AAV vector of the present invention comprises construct 100ATG short bGH polyA sequence set forth in SEQ ID NO: 11.

[0028] In a fourth aspect, the AAV vector of the present invention comprises construct 103ATG comprising the nucleic acid sequence set forth in SEQ ID NO: 12.

[0029] In the fifth aspect, the AAV vector of the present invention includes a construct 103ATG short bGH polyA containing the nucleic acid sequence set forth in SEQ ID NO: 13.

[0030] In the sixth aspect, the AAV vector of the present invention includes a construct 105ATG bGH polyA containing the nucleic acid sequence set forth in SEQ ID NO: 14.

[0031] In the seventh aspect, the AAV vector of the present invention includes a construct DC172ATG FVIII containing the nucleic acid sequence set forth in SEQ ID NO: 15.

[0032] In the eighth aspect, the AAV vector of the present invention includes a construct DC172ATG FVIII hAAT containing the nucleic acid sequence set forth in SEQ ID NO: 16.

[0033] In the ninth aspect, the AAV vector of the present invention includes a construct DC172 2×HCR ATG FVIII containing the nucleic acid sequence set forth in SEQ ID NO: 17.

[0034] In the tenth aspect, the AAV vector of the present invention includes a construct DC172 2×HCR ATG FVIII hAAT containing the nucleic acid sequence set forth in SEQ ID NO: 18.

[0035] In the eleventh aspect, the AAV vector of the present invention includes a construct 2×serpin A hAAT ATG FVIII containing the nucleic acid sequence set forth in SEQ ID NO: 19.

[0036] In the twelfth aspect, the AAV vector of the present invention includes a construct 2×serpin A hAAT ATG FVIII 2×μ-globulin enhancer containing the nucleic acid sequence set forth in SEQ ID NO: 20.

[0037] In the thirteenth aspect, the AAV vector of the present invention a construct 100ATG short polyA 2×μ-globulin enhancer containing the nucleic acid sequence set forth in SEQ ID NO: 21.

[0038] In the 14th aspect, the AAV vector of the present invention comprises the construct Factor VIII - BMN001 containing the nucleic acid sequence set forth in SEQ ID NO: 22.

[0039] In the 15th aspect, the AAV vector of the present invention comprises the construct Factor VIII - BMN002 sequence set forth in SEQ ID NO: 23.

[0040] In the 16th aspect, the AAV vector of the present invention comprises the construct 99 containing the nucleic acid sequence set forth in SEQ ID NO: 24.

[0041] In the 17th aspect, the AAV vector of the present invention comprises the construct 100 containing the nucleic acid sequence set forth in SEQ ID NO: 25.

[0042] In the 18th aspect, the AAV vector of the present invention comprises the construct 100 reverse orientation containing the nucleic acid sequence set forth in SEQ ID NO: 26.

[0043] In the 19th aspect, the AAV vector of the present invention is the construct 100AT containing the nucleic acid sequence set forth in SEQ ID NO: 27.

[0044] In the 20th aspect, the AAV vector of the present invention is the construct 100AT 2×MG containing the nucleic acid sequence set forth in SEQ ID NO: 28.

[0045] In the 21st aspect, the AAV vector of the present invention comprises the construct 100AT 2×MG bGH polyA containing the nucleic acid sequence set forth in SEQ ID NO: 29.

[0046] In the 22nd aspect, the AAV vector of the present invention comprises the construct 100AT 2×MG (reverse) bGH polyA containing the nucleic acid sequence set forth in SEQ ID NO: 30.

[0047] In the 23rd aspect, the AAV vector of the present invention comprises the construct 100 bGH polyA containing the nucleic acid sequence set forth in SEQ ID NO: 31.

[0048] In the 24th aspect, the AAV vector of the present invention comprises constructs 100 to 400 containing the nucleic acid sequence set forth in SEQ ID NO: 32.

[0049] In the 25th aspect, the AAV vector of the present invention comprises construct 101 containing the nucleic acid sequence set forth in SEQ ID NO: 33.

[0050] In the 26th aspect, the AAV vector of the present invention comprises the construct 102 sequence containing the nucleic acid sequence set forth in SEQ ID NO: 34.

[0051] In the 27th aspect, the AAV vector of the present invention comprises construct 103 containing the nucleic acid sequence set forth in SEQ ID NO: 35.

[0052] In the 29th aspect, the AAV vector of the present invention comprises the construct 103 reverse orientation containing the nucleic acid sequence set forth in SEQ ID NO: 36.

[0053] In the 30th aspect, the AAV vector of the present invention comprises construct 103AT containing the nucleic acid sequence set forth in SEQ ID NO: 37.

[0054] In the 31st aspect, the AAV vector of the present invention comprises construct 103AT 2×MG containing the nucleic acid sequence set forth in SEQ ID NO: 38.

[0055] In the 32nd aspect, the AAV vector of the present invention comprises construct 103AT 2×MG bGH polyA containing the nucleic acid sequence set forth in SEQ ID NO: 39.

[0056] In the 33rd aspect, the AAV vector of the present invention comprises construct 103 bGH polyA containing the nucleic acid sequence set forth in SEQ ID NO: 40.

[0057] In the 34th aspect, the AAV vector of the present invention comprises construct 104 containing a nucleic acid containing the nucleic acid sequence set forth in SEQ ID NO: 41.

[0058] In the 35th aspect, the AAV vector of the present invention includes a construct 105 containing the nucleic acid sequence set forth in SEQ ID NO: 42.

[0059] In the 36th aspect, the AAV vector of the present invention includes a construct 106 containing the nucleic acid sequence set forth in SEQ ID NO: 43.

[0060] In the 37th aspect, the AAV vector of the present invention includes a construct 106AT containing the nucleic acid sequence set forth in SEQ ID NO: 44.

[0061] In the 38th aspect, the AAV vector of the present invention includes a construct 2×Serpin A hAAT containing the nucleic acid sequence set forth in SEQ ID NO: 45.

[0062] In still other embodiments, the present invention relates to a vector construct encoding a functional Factor VIII polypeptide, said construct comprising one or more of the individual components of the above constructs and their combinations in one or more different orientations. The present invention also relates to the above constructs in reverse orientation.

[0063] The single-stranded AAV vector of the present invention is less than about 7.0 kb in length, or less than 6.5 kb in length, or less than 6.4 kb in length, or less than 6.3 kb in length, or less than 6.2 kb in length, or less than 6.0 kb in length, or less than 5.8 kb in length, or less than 5.6 kb in length, or less than 5.5 kb in length, or less than 5.4 kb in length, or less than 5.4 kb in length, or less than 5.2 kb in length or less than 5.0 kb in length. The single-stranded AAV vector of the present invention is in the range of about 5.0 kb to about 6.5 kb in length, about 4.8 kb to about 5.2 kb in length, or 4.8 kb to 5.3 kb in length, or about 4.9 kb to about 5.5 kb in length, or about 4.8 kb to about 6.0 kb in length, or about 5.0 kb to 6.2 kb in length or about 5.1 kb to about 6.3 kb in length, or about 5.2 kb to about 6.4 kb in length, or about 5.5 kb to about 6.5 kb in length.

[0064] In another embodiment, the present invention provides a method for producing recombinant adeno-associated virus (AAV) particles comprising any of the AAV vectors of the present invention. The method includes culturing cells transfected with any of the AAV vectors of the present invention, and recovering recombinant AAV from the supernatant of the transfected cells.

[0065] The cells of the present invention are any cell type susceptible to baculovirus infection, such as insect cells such as High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38. Preferred mammalian cells that can be used are HEK293, HeLa, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT1080, A549, Cos-7, ARPE-19 and MRC-5 cells, for example, mammalian cells such as HEK293, HeLa, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT1080, A549, Cos-7, ARPE-19 and MRC-5 cells.

[0066] The present invention also provides virus particles comprising any of the AAV vectors of the present invention or any virus particles produced by the aforementioned method of the present invention.

[0067] "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to at least one AAV capsid tan Refers to virus particles composed of a protein and a polynucleotide AAV vector that has formed a capsid. When the said particles contain a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene intended to be delivered to mammalian cells), they are usually referred to as "AAV vector particles" or simply "AAV vectors". Thus, since such vectors are contained within AAV vector particles, the production of AAV vector particles necessarily includes the production of AAV vectors.

[0068] The present invention also provides cells containing any of the AAV vectors of the present invention, and virus particles produced by these cells of the present invention.

[0069] In another embodiment, the present invention provides a method for treating a patient suffering from hemophilia A, the method comprising administering to the patient an effective amount of any of the AAV vectors of the present invention, or virus particles produced by the virus particles of the present invention or the method of the present invention.

[0070] In a further embodiment, the present invention provides the use of any of the AAV vectors of the present invention for preparing a medicament for the treatment of hemophilia A. In one aspect, the said medicament contains an amount of AAV vector that expresses human FVIII in an amount effective for treating hemophilia A.

[0071] In another embodiment, the present invention provides a composition containing any of the AAV vectors of the present invention for the treatment of hemophilia A. In one aspect, the said composition contains an amount of AAV vector that expresses human FVIII in an amount effective for treating hemophilia A.

[0072] In another embodiment, the AAV vector of the present invention is used to produce AAV virus particles useful for treating a patient suffering from hemophilia A. In certain embodiments, for example, the following are provided: (Item 1) An adeno-associated virus (AAV) factor VIII (FVIII) vector comprising a nucleic acid containing an AAV2 5' inverted terminal repeat (ITR), a liver-specific transcriptional regulatory region, an optimally codon-functional active FVIII coding region, one or more introns as desired, a polyadenylation sequence, and an AAV2 3' ITR. (Item 2) The AAV according to item 1, wherein the nucleic acid comprises a nucleotide sequence selected from the group consisting of FVIII vector, i. The Proto1 sequence set forth in SEQ ID NO: 1, ii. The Proto1S sequence set forth in SEQ ID NO: 2, iii. The Proto2S sequence set forth in SEQ ID NO: 3, iv. The Proto3S sequence set forth in SEQ ID NO: 4, v. The Proto4 sequence set forth in SEQ ID NO: 5, vi. The Proto5 sequence set forth in SEQ ID NO: 6, vii. The Proto6 sequence set forth in SEQ ID NO: 7, viii. The Proto7 sequence set forth in SEQ ID NO: 8, ix. The construct 100ATG sequence set forth in SEQ ID NO: 9, x. The construct 100ATG bGH polyA sequence set forth in SEQ ID NO: 10, xi. The construct 100ATG short bGH polyA sequence set forth in SEQ ID NO: 11, xii. The construct 103ATG sequence set forth in SEQ ID NO: 12, xiii. The construct 103ATG short bGH polyA sequence set forth in SEQ ID NO: 13, xiv. The construct 105ATG bGH polyA sequence set forth in SEQ ID NO: 14, xv. The construct DC172ATG FVIII sequence set forth in SEQ ID NO: 15, xvi. The construct DC172ATG FVIII hAAT sequence set forth in SEQ ID NO: 16. xvii. The construct DC172 2×HCR ATG FVIII sequence, xviii. The construct DC172 2×HCR ATG FVIII hAAT sequence described in SEQ ID NO: 18, xix. The construct 2×Serpin A hAAT ATG described in SEQ ID NO: 19 FVIII sequence, xx. The construct 2×Serpin A hAAT ATG FVIII 2×μ-globulin enhancer sequence described in SEQ ID NO: 20, xxi. The construct 100ATG short polyA 2×μ-globulin enhancer sequence described in SEQ ID NO: 21, xxii. The construct Factor VIII - BMN001 sequence described in SEQ ID NO: 22, xxiii. The construct Factor VIII - BMN002 sequence described in SEQ ID NO: 23, xxiv. The construct 99 sequence described in SEQ ID NO: 24, xxv. The construct 100 sequence described in SEQ ID NO: 25, xxvi. The construct 100 reverse orientation sequence described in SEQ ID NO: 26, xxvii. The construct 100AT sequence described in SEQ ID NO: 27, xxviii. The construct 100AT 2×MG sequence described in SEQ ID NO: 28, xxix. The construct 100AT 2×MG polyA sequence described in SEQ ID NO: 29, xxx. The construct 100AT 2×MG (reverse) bGH polyA sequence described in SEQ ID NO: 30, xxxi. The construct 100 bGH polyA sequence described in SEQ ID NO: 31, xxxii. The construct 100 - 400 sequence described in SEQ ID NO: 32, xxxiii. The construct 101 sequence described in SEQ ID NO: 33, xxxiv. The construct 102 sequence described in SEQ ID NO: 34, xxxv. The construct 103 sequence described in SEQ ID NO: 35, xxxvi. The construct 103 reverse orientation array described in SEQ ID NO: 36, xxxvii. The construct 103 AT array described in SEQ ID NO: 37, xxxviii. The construct 103 AT 2×MG array described in SEQ ID NO: 38, xxxix. The construct 103 AT 2×MG polyA array described in SEQ ID NO: 39, xl. The construct 103 bGH polyA array described in SEQ ID NO: 40, xli. The construct 104 array described in SEQ ID NO: 41, xlii. The construct 105 array described in SEQ ID NO: 42, xliii. The construct 106 array described in SEQ ID NO: 43, xliv. The construct 106 AT array described in SEQ ID NO: 44, and xlv. The construct 2×serpin A hAAT array described in SEQ ID NO: 45. (Item 3) A method for producing recombinant adeno-associated virus (AAV) particles, comprising: A) culturing cells transfected with the AAV vector according to Item 1 or 2; B) recovering recombinant AAV particles from the supernatant of the transfected cells. The above method. (Item 4) Viral particles comprising the viral vector according to Item 1 or 2. (Item 5) Cells comprising the viral vector according to Item 1 or 2. (Item 6) A method for treating a patient suffering from hemophilia A, comprising administering to the patient an effective amount of the AAV FVIII vector according to Item 1 or 2. (Item 7) Use of the AAV FVIII vector according to Item 1 or 2 for preparing a medicament for the treatment of hemophilia A. (Item 8) A composition for the treatment of hemophilia A, comprising the AAV FVIII vector according to item 1 or 2.

Brief Description of Drawings

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Mode for Carrying Out the Invention

[0074] The large-capacity AAV vector is randomly cleaved at the 5' end and lacks the 5' AAV ITR. Since AAV is a single-stranded DNA virus and packages either the sense strand or the antisense strand, the sense strand in the large-capacity AAV vector lacks the 5' AAV ITR and may lack a part of the 5' end of the gene encoding the target protein. The antisense strand in the large-capacity AAV vector lacks the 3' ITR and may lack a part of the 3' end of the gene encoding the target protein. By annealing the sense and antisense cleaved genomes in target cells, a functional transgene is produced in large-capacity AAV vector-infected cells.

[0075] The present invention provides an AAV vector encoding a functionally active FVIII, i.e., a fully packaged AAV FVIII vector or an AAV FVIII vector having high expression activity. The AAV FVIII vector of the present invention has improved expression / particles, improved AAV virus production efficiency, and simplified purification. Expression is enhanced by introducing one or more introns into the FVIII protein coding region. Expression is also enhanced by resetting the number and positioning of enhancers.

[0076] UCL SQ vector The UCL SQ vector is described in detail in U.S. Patent Application Publication No. 2013 / 0024960A1, published on January 24, 2013 by Nathwani et al. (which is hereby incorporated by reference in its entirety), and McIntosh etal., Blood 121:3335-3344, 2013 However, this vector is a high-capacity, i.e., greater than 5.0 kb, AAV vector. As shown in Figure 1, the UCL SQ vector, from left to right, contains the AAV serotype 2 (AAV2) 5’ ITR, wild-type AAV2 viral sequence, 34-base human apolipoprotein E (ApoE) / C1 enhancer, 32-base human alpha-1 antitrypsin (AAT) promoter distal X region, 186-base human AAT promoter (including a 42-base 5’ untranslated region (UTR) sequence), a human FVIII sequence with codons optimized such that the B domain is replaced with a 14-amino acid SQ sequence, a 49-base synthetic polyadenylation sequence, wild-type AAV2 viral sequence, and the AAV2 3’ ITR. The UCL SQ vector is 5081 bases in length.

[0077] As shown in Nathwani et al., U.S. Patent Application Publication No. 2013 / 0024960A1, published January 24, 2013, and McIntosh et al., Blood 121:3335-3344, 2013, the UCL SQ vector expresses functionally active FVIII in vitro and in vivo.

[0078] Proto1 vector, Proto1S vector, Proto2S vector and Proto3S vector To avoid problems with high-capacity AAV vectors and / or to increase the expression of AAV vectors, the present invention provides a fully packaged, smaller, i.e., less than 5.0 kb, AAV vector encoding an FVIII SQ variant. The 4970 bp nucleotide sequence of the Proto1 sequence is set forth in SEQ ID NO: 1.

[0079] To generate the AAV vector Proto1, sequences that were thought to be unnecessary for the production of functionally active FVIII were removed compared to the UCL SQ vector. As shown in Example 1, 110 bases of foreign DNA, including 53 bases of the AAV2 viral sequence on the 3'-side of the AAV2 5' ITR, 46 bases of the AAV2 viral sequence on the 5'-side of the AAV2 3' ITR, and 11 bases adjacent to the codon-optimized FVIII SQ coding region, were removed. The resulting Proto1 vector is 4970 bases in length. When designed, it was unclear whether the Proto1 vector could express functional FVIII polypeptide in vitro or in vivo.

[0080] To generate the AAV vector Proto1S, 10 bases at the 3'-end of the AAV2 5' ITR and 10 bases at the 5'-end of the AAV32 3' ITR were removed from the Proto1 vector. The resulting Proto1S vector is 4950 bases in length. The nucleotide sequence of the Proto1S sequence is set forth in SEQ ID NO: 2.

[0081] To generate the AAV vector Proto2S, a synthetic 100-base intron was inserted between exon 1 and exon 2 of the codon-optimized FVIII SQ sequence in the Proto1S vector. A 34-base ApoE / C1 enhancer and a 32-base human AAT promoter distal X region were removed from upstream of the human AAT promoter and inserted in reverse orientation (compared to the orientation when these elements are located upstream of the human AAT promoter) within the synthetic intron. The resulting Proto2S vector is 4983 bases in length. The nucleotide sequence of the Proto2S sequence is set forth in SEQ ID NO: 3.

[0082] To generate the AAV vector Proto3S, the human AAT promoter distal X region was removed from the Proto2S vector and replaced with a second copy of the 34-base ApoE / C1 enhancer in reverse orientation. The resulting Proto3S vector is 4984 bases in length. The nucleotide sequence of the Proto3S sequence is set forth in SEQ ID NO: 4.

[0083] Proto4 vector, ProtoS vector, Proto6 vector and Proto7 vector To reduce the size of the AAV vector and / or increase the expression of the AAV vector, the present invention also provides an AAV vector that encodes a fully packaged, small, i.e., less than 5.0 kb, B domain and a3 domain deleted FVIII.

[0084] To generate the AAV vector Proto4, a 14 amino acid SQ sequence and the a3 domain located adjacent to the C domain were removed from the Proto1 vector. The total amount of the removed FVIII sequence is 55 amino acids or 165 bases. The resulting Proto4 vector is 4805 bases in length. The nucleotide sequence of the Proto4 sequence is set forth in SEQ ID NO: 5.

[0085] To generate the AAV vector Proto5, a 129 base truncated FVIII intron was inserted between exon 1 and exon 2 of the FVIII sequence with optimized codons in the Proto4 vector. The resulting Proto5 vector is 4934 bases in length. The nucleotide sequence of the Proto5 sequence is set forth in SEQ ID NO: 6.

[0086] To generate the AAV Proto6 vector, a 34 base FVIII intron was replaced with a second copy of the 34 base human ApoE / C1 enhancer in the forward orientation in the Proto5 vector. The resulting Proto6 vector is 4934 bases in length. The nucleotide sequence of the Proto6 sequence is set forth in SEQ ID NO: 7.

[0087] To generate the AAV Proto7 vector, a 34 base FVIII intron was replaced with a second copy of the 34 base human ApoE / C1 enhancer in the reverse orientation in the Proto5 vector. The resulting Proto7 vector is 4934 bases in length. The nucleotide sequence of the Proto7 sequence is set forth in SEQ ID NO: 8.

[0088] Additional AAV FVIII vectors with an upward promoter / enhancer sequence

[0089] To increase the expression of B domain- and a3 domain-deleted FVIII, large-capacity AAV vectors with strong promoters were generated, and these constructs were generated with modified enhancer sequences and / or promoter sequences. In some embodiments, the AAV FVIII vectors express truncated functional FVIII. These constructs include one or more promoter sequences and enhancer sequences such as ApoE HCR or a fragment thereof, μ-globulin enhancer or a fragment thereof, human α1-antitrypsin promoter (hAAT) or a fragment thereof, serpin A enhancer or a fragment thereof, LP1 promoter enhancer or a fragment thereof, or macroglobulin enhancer or a fragment thereof. These constructs include polyadenylation sequences such as the bGH polyA sequence or the synthetic rabbit β-globin polyA sequence. In some embodiments, the construct includes an intron or a fragment of an intron such as the hAAT intron or the human β-globin intron.

[0090] Construct 100ATG is 5511 bases in length. This construct is described in SEQ ID NO: 9, where bases 1-145 are the 5’ AAV2 ITR, bases 160-502 are ApoE HCR, bases 509-726 are the hAAT promoter, bases 727-910 are the modified human β-globin second intron, bases 923-5296 are codon-optimized SQ FVIII, bases 5305-5352 are the synthetic rabbit β-globin polyA, and bases 5367-5511 are the 3’ AAV2 ITR.

[0091] Construct 100ATG bGH polyA is 5688 bases long. This construct is described in SEQ ID NO: 10, where bases 1-145 are the 5’ AAV2 ITR, bases 160-502 are the ApoE HCR, bases 509-726 are the hAAT promoter, bases 727-910 are the modified human β-globin intron 2, bases 923-5296 are codon-optimized SQ FVIII, bases 5305-5529 are the bGH polyA, and bases 5544-5688 are the 3’ AAV2 ITR.

[0092] Construct 100ATG short bGH polyA is 5613 bases long. This construct is described in SEQ ID NO: 11, where bases 1-145 are the 5’ AAV2 ITR, bases 160-502 are the ApoE HCR, bases 509-726 are the hAAT promoter, bases 727-910 are the modified human β-globin intron 2, bases 923-5296 are codon-optimized SQ FVIII, bases 5305-5454 are the short bGH polyA, and bases 5469-5613 are the 3’ AAV2 ITR.

[0093] Construct 103ATG is 5362 bases long. This construct is described in SEQ ID NO: 12, where bases 1-145 are the 5’ AAV2 ITR, bases 169-344 are four copies of the 44bp ApoE repeat, bases 360-577 are the hAAT promoter, bases 578-761 are the modified human β-globin intron 2, bases 774-5147 are codon-optimized SQ FVIII, bases 5156-5203 are the synthetic rabbit β-globin polyA, and bases 5218-5362 are the 3’ AAV2 ITR.

[0094] Construct 103ATG short bGH polyA is 5,464 bases long. This construct is described in SEQ ID NO: 13, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 344 are 4 copies of the 44bp ApoE repeat, bases 360 to 577 are the hAAT promoter, bases 578 to 761 are the modified human β-globin intron 2, bases 774 to 5,147 are codon-optimized SQ FVIII, bases 5,156 to 5,305 are the bGH short polyA, and bases 5,320 to 5,464 are the 3’ AAV2 ITR.

[0095] Construct 105ATG bGH polyA is 6,354 bases long. This construct is described in SEQ ID NO: 14, where bases 1 to 145 are the 5’ AAV2 ITR, bases 173 to 512 are 2 copies (2×) of the 170bp microglobulin enhancer, bases 519 to 736 are the hAAT promoter, bases 737 to 920 are the modified human β-globin intron 2, bases 933 to 5,306 are codon-optimized SQ FVIII, bases 5,315 to 5,539 are the bGH polyA, bases 5,546 to 6,195 are 2 copies (2×) of the 325bp ApoE HCR, and bases 6,210 to 6,354 are the 3’ AAV2 ITR.

[0096] Construct DC172ATG FVIII is 6,308 bases long. This construct is described in SEQ ID NO: 15, where bases 1 to 145 are the 5’ AAV2 ITR, bases 160 to 449 are 2 copies (2×) of the 145bp macroglobulin enhancer, bases 450 to 1,347 are the 898bp hAAT promoter, bases 1,348 to 1,531 are the modified human β-globin intron 2, bases 1,544 to 5,917 are codon-optimized SQ FVIII, bases 5,926 to 6,149 are the bGH polyA, and bases 6,164 to 6,308 are the 3’ AAV2 ITR.

[0097] Construct DC172ATG FVIII hAAT is 5,635 bases long. This construct is described as SEQ ID NO: 16, where bases 1 to 145 are 5’ AAV2 It is an ITR. Bases 160 to 449 are 2 copies (2×) of a 145 bp macroglobulin enhancer, bases 457 to 674 are the hAAT promoter, bases 675 to 858 are the modified human β-globin intron 2, bases 871 to 5244 are codon-optimized SQ FVIII, bases 5253 to 5476 are bGH polyA, and bases 5490 to 5635 are the 3’ AAV2 ITR.

[0098] Construct DC172 2×HCR ATG FVIII is 6962 bases in length. This construct is described in SEQ ID NO: 17, and bases 1 to 145 are the 5’ AAV2 ITR. Bases 160 to 807 are 2 copies (2×) of a 321 bp ApoE HCR, bases 814 to 1103 are 2 copies (2×) of a 145 bp macroglobulin enhancer, bases 1104 to 2001 are an 898 bp hAAT promoter, bases 2002 to 2185 are the modified human β-globin intron 2, bases 2198 to 6571 are codon-optimized SQ FVIII, bases 6580 to 6803 are bGH polyA, and bases 6818 to 6962 are the 3’ AAV2 ITR.

[0099] Construct DC172 2×HCR ATG FVIII hAAT is 6289 bases in length. This construct is described in SEQ ID NO: 18, and bases 1 to 145 are the 5’ AAV2 ITR, bases 160 to 807 are 2 copies (2×) of a 321 bp ApoE HCR, bases 814 to 1103 are 2 copies (2×) of a 145 bp macroglobulin enhancer, bases 1111 to 1328 are the hAAT promoter, bases 1329 to 1512 are the modified human β-globin intron 2, bases 1525 to 5898 are codon-optimized SQ FVIII, bases 5907 to 6130 are bGH polyA, and bases 6245 to 6289 are the 3’ AAV2 ITR.

[0100] The construct 2×Serpin A hAAT ATG FVIII is 5430 bases long. This construct is described in SEQ ID NO: 19, where bases 1 to 145 are the 5’ AAV2 ITR, bases 168 to 309 are two copies (2×) of the 71bp Serpin A enhancer, bases 326 to 543 are the hAAT promoter, bases 544 to 727 are the modified human β-globin intron 2, bases 740 to 5113 are codon-optimized SQ FVIII, bases 5122 to 5271 are the short bGH polyA, and bases 5286 to 5430 are the 3’ AAV2 ITR.

[0101] The construct 2×Serpin A hAAT ATG FVIII 2×μ-globulin enhancer is 5779 bases long. This construct is described in SEQ ID NO: 20, where bases 1 to 145 are the 5’ AAV2 ITR, bases 168 to 309 are two copies (2×) of the 71bp Serpin A enhancer, bases 326 to 543 are the hAAT promoter, bases 544 to 727 are the modified human β-globin intron 2, bases 740 to 5113 are codon-optimized SQ FVIII, bases 5122 to 5271 are the short bGH polyA, bases 5279 to 5618 are two copies (2×) of the 170bp μ-globulin enhancer, and bases 5635 to 5779 are the 3’ AAV2 ITR.

[0102] The construct 100ATG short bGH polyA 2×μ-globulin enhancer is 5962 bases long. This construct is described in SEQ ID NO: 21, where bases 1 to 145 are the 5’ AAV2 ITR, bases 160 to 502 are the ApoE HCR, bases 509 to 726 are the hAAT promoter, bases 727 to 910 are the modified human β-globin intron 2, bases 923 to 5296 are codon-optimized SQ FVIII, bases 5305 to 5454 are the short bGH polyA, bases 5462 to 5801 are two copies (2×) of the 170bp microglobulin enhancer, and bases 5818 to 5962 are the 3’ AAV2 ITR.

[0103] The construct Factor VIII - BMN001 is 5919 bases in length. This construct is described in SEQ ID NO: 22, where bases 1 - 145 are the 5’ AAV2 ITR, bases 160 - 480 are the ApoE HCR, bases 487 - 884 are the 398bp hAAT promoter, bases 885 - 1145 are the truncated hAAT intron, bases 1155 - 5528 are the codon - optimized SQ FVIII, bases 5537 - 5760 are the bGH polyA, and bases 5775 - 5919 are the 3’ AAV2 ITR.

[0104] The construct FVIII - BMN002 is 5306 bases in length. This construct is described in SEQ ID NO: 23, where bases 1 - 145 are the 5’ AAV2 ITR, bases 175 - 705 are the LP1 promoter / enhancer, bases 718 - 5091 are the codon - optimized SQ FVIII, bases 5100 - 5147 are the synthetic rabbit β - globin polyA, and bases 5162 - 5306 are the 3’ AAV2 ITR.

[0105] The construct 99 is 5461 bases in length. This construct is described in SEQ ID NO: 24, where bases 1 - 145 are the 5’ AAV2 ITR, bases 169 - 627 are the ApoE HCR / MAR, bases 634 - 866 are the hAAT promoter, bases 873 - 5246 are the codon - optimized SQ FVIII, bases 5255 - 5302 are the synthetic rabbit β - globin polyA, and bases 5317 - 5461 are the 3’ AAV2 ITR.

[0106] The construct 100 is 5327 bases in length. This construct is described in SEQ ID NO: 25, where bases 1 - 145 are the 5’ AAV2 ITR, bases 169 - 493 are the ApoE HCR, bases 509 - 726 are the hAAT promoter, bases 739 - 5112 are the codon - optimized SQ FVIII, bases 5121 - 5168 are the synthetic rabbit β - globin polyA, and bases 5183 - 5327 are the 3’ AAV2 ITR.

[0107] The construct 100 reverse orientation is 5309 bases in length. This construct is described in SEQ ID NO: 26, where bases 1 to 145 are the 5’ AAV2 ITR, bases 160 to 484 are the reverse orientation ApoE HCR, bases 491 to 708 are the hAAT promoter, bases 721 to 5094 are the codon-optimized SQ FVIII, bases 5103 to 5150 are the synthetic rabbit β-globin polyA, and bases 5165 to 5309 are the 3’ AAV2 ITR.

[0108] The construct 100 AT is 5532 bases in length. This construct is described in SEQ ID NO: 27, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 493 are the ApoE HCR, bases 509 to 726 are the hAAT promoter, bases 727 to 931 are the hAAT intron, bases 944 to 5317 are the codon-optimized SQ FVIII, bases 5326 to 5373 are the synthetic rabbit β-globin polyA, and bases 5388 to 5532 are the 3’ AAV2 ITR.

[0109] The construct 100 AT 2×MG is 5877 bases in length. This construct is described in SEQ ID NO: 28, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 493 are the ApoE HCR, bases 508 to 847 are two copies (2×) of the 170bp μ-globulin enhancer, bases 854 to 1071 are the hAAT promoter, bases 1072 to 1276 are the hAAT intron, bases 1289 to 5662 are the codon-optimized SQ FVIII, bases 5671 to 5718 are the synthetic rabbit β-globin polyA, and bases 5733 to 5877 are the 3’ AAV2 ITR.

[0110] Construct 100AT 2×MG bGH polyA is 6,054 bases long. This construct is described in SEQ ID NO: 29, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 493 are the ApoE HCR, bases 508 to 847 are two copies (2×) of the 170bp μ-globulin enhancer, bases 854 to 1,071 are the hAAT promoter, bases 1,072 to 1,276 are the hAAT intron, bases 1,289 to 5,662 are codon-optimized SQ FVIII, bases 5,671 to 5,895 are the bGH polyA, and bases 5,910 to 6,054 are the 3’ AAV2 ITR.

[0111] Construct 100AT 2×MG (reverse) bGH polyA is 6,054 bases long. This construct is described in SEQ ID NO: 30, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 493 are the ApoE HCR, bases 508 to 847 are two copies (2×) of the reverse-oriented 170bp μ-globulin enhancer, bases 854 to 1,071 are the hAAT promoter, bases 1,072 to 1,276 are the hAAT intron, bases 1,289 to 5,662 are codon-optimized SQ FVIII, bases 5,671 to 5,895 are the bGH polyA, and bases 5,910 to 6,054 are the 3’ AAV2 ITR.

[0112] Construct 100bGH polyA is 5,504 bases long. This construct is described in SEQ ID NO: 31, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 493 are the ApoE HCR, bases 509 to 726 are the hAAT promoter, bases 739 to 5,112 are codon-optimized SQ FVIII, base pairs 5,121 to 5,345 are the bGH polyA, and bases 5,360 to 5,504 are the 3’ AAV2 ITR.

[0113] Constructs 100 to 400 are 5507 bases in length. This construct is described in SEQ ID NO: 32, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 493 are the ApoE HCR, bases 512 to 906 are the 398bp hAAT promoter, bases 919 to 5292 are the codon-optimized SQ FVIII, bases 5301 to 5348 are the synthetic rabbit β-globin polyA, and bases 5363 to 5507 are the 3’ AAV2 ITR.

[0114] Construct 101 is 5311 bases in length. This construct is described in SEQ ID NO: 33, where bases 1 to 145 are the 5’ AAV2 ITR, bases 170 to 477 are two copies (2×) of the 154bp ApoE HCR, bases 493 to 710 are the hAAT promoter, bases 723 to 5096 are the codon-optimized SQ FVIII, bases 5105 to 5152 are the synthetic rabbit β-globin polyA, and bases 5167 to 5311 are the 3’ AAV2 ITR.

[0115] Construct 102 is 5156 bases in length. This construct is described in SEQ ID NO: 34, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 322 are the 154bp ApoE HCR, bases 338 to 555 are the hAAT promoter, bases 568 to 4941 are the codon-optimized SQ FVIII, bases 4950 to 4997 are the synthetic rabbit β-globin polyA, and bases 5012 to 5156 are the 3’ AAV2 ITR.

[0116] Construct 103 is 5178 bases in length. This construct is described in SEQ ID NO: 35, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 344 are four copies (4x) of the 44bp ApoE HCR, bases 360 to 577 are the hAAT promoter, bases 590 to 4963 are the codon-optimized SQ FVIII, bases 4972 to 5019 are the synthetic rabbit β-globin polyA, and bases 5034 to 5178 are the 3’ AAV2 ITR.

[0117] The construct 103 reverse orientation is 5160 bases long. This construct is described in SEQ ID NO: 36, where bases 1 to 145 are the 5’ AAV2 ITR, bases 160 to 335 are 4 copies (4x) of the reverse orientation 44bp ApoE HCR, bases 342 to 559 are the hAAT promoter, bases 572 to 4945 are codon-optimized SQ FVIII, bases 4954 to 5001 are the synthetic rabbit β-globin polyA, and bases 5016 to 5160 are the 3’ AAV2 ITR.

[0118] The construct 103 AT is 5383 bases long. This construct is described in SEQ ID NO: 37, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 344 are 4 copies (4x) of the 44bp ApoE HCR, bases 360 to 577 are the hAAT promoter, bases 578 to 782 are the hAAT intron, bases 795 to 4374 are codon-optimized SQ FVIII, bases 5177 to 5224 are the synthetic rabbit β-globin polyA, and bases 5239 to 5383 are the 3’ AAV2 ITR.

[0119] The construct 103 AT 2×MG is 5728 bases long. This construct is described in SEQ ID NO: 38, where bases 1 to 145 are the 5’ AAV2 ITR, bases 169 to 344 are 4 copies (4x) of the 44bp ApoE HCR, bases 359 to 698 are 2 copies (2×) of the 170bp μ-globulin enhancer, bases 705 to 922 are the hAAT promoter, bases 923 to 1127 are the hAAT intron, bases 1140 to 5513 are codon-optimized SQ FVIII, bases 5522 to 5569 are the synthetic rabbit β-globin polyA, and bases 5584 to 5728 are the 3’ AAV2 ITR.

[0120] Construct 103 AT 2×MG bGH polyA is 5905 bases long. This construct is described in SEQ ID NO: 39. Bases 1 to 145 are 5’ AAV2 ITR. Bases 169 to 344 are 4 copies (4x) of 44bp ApoE HCR. Bases 359 to 698 are 2 copies (2×) of 170bp μ-globulin enhancer. Bases 705 to 922 are hAAT promoter. Bases 923 to 1127 are hAAT intron. Bases 1140 to 5513 are codon-optimized SQ FVIII. Bases 5522 to 5746 are synthetic rabbit β-globin polyA. Bases 5761 to 5905 are 5’ AAV2 ITR.

[0121] Construct 103 bGH polyA is 5355 bases long. This construct is described in SEQ ID NO: 40. Bases 1 to 145 are 5’ AAV2 ITR. Bases 169 to 344 are 4 copies (4x) of 44bp ApoE HCR. Bases 360 to 577 are hAAT promoter. Bases 590 to 4963 are codon-optimized SQ FVIII. Bases 4972 to 5196 are synthetic rabbit β-globin polyA. Bases 5211 to 5355 are 3’ AAV2 ITR.

[0122] Construct 104 is 5618 bases long. This construct is described in SEQ ID NO: 41. Bases 1 to 145 are 5’ AAV2 ITR. Bases 169 to 784 are 4 copies (4x) of 154bp ApoE HCR. Bases 800 to 1017 are hAAT promoter. Bases 1030 to 5403 are codon-optimized SQ FVIII. Bases 5412 to 5459 are synthetic rabbit β-globin polyA. Bases 5474 to 5618 are 3’ AAV2 ITR.

[0123] Construct 105 is 5993 bases in length. This construct is described in SEQ ID NO: 42, where bases 1 to 145 are the 5’ AAV2 ITR, bases 173 to 512 are two copies (2×) of the 170 bp μ-globulin enhancer, bases 519 to 736 are the hAAT promoter, bases 749 to 5122 are codon-optimized SQ FVIII, bases 5131 to 5178 are the synthetic rabbit β-globin polyA, bases 5185 to 5834 are two copies (2×) of the ApoE HCR, and bases 5849 to 5993 are the 3’ AAV2 ITR.

[0124] Construct 106 is 5337 bases in length. This construct is described in SEQ ID NO: 43, where bases 1 to 145 are the 5’ AAV2 ITR, bases 173 to 512 are two copies (2×) of the 170 bp μ-globulin enhancer, bases 519 to 736 are the hAAT promoter, bases 749 to 5122 are codon-optimized SQ FVIII, bases 5131 to 5178 are the synthetic rabbit β-globin polyA, and bases 5193 to 5337 are the 3’ AAV2 ITR.

[0125] Construct 106AT is 5542 bases in length. This construct is described in SEQ ID NO: 44, where bases 1 to 145 are the 5’ AAV2 ITR, bases 173 to 512 are two copies (2×) of the 170 bp μ-globulin enhancer, bases 519 to 736 are the hAAT promoter, bases 737 to 941 are the hAAT intron, bases 954 to 5327 are codon-optimized SQ FVIII, bases 5336 to 5383 are the synthetic rabbit β-globin polyA, and bases 5398 to 5542 are the 3’ AAV2 ITR.

[0126] Construct 2x SerpinA hAAT is 5126 bases. This construct is set forth in SEQ ID NO:45, where bases 1-145 are the 5' AAV2 ITR, bases 160-301 are the ApoE HCR, bases 308-525 are the hAAT promoter, bases 538-4911 are codon-optimized SQ FVIII, bases 4920-4967 are synthetic rabbit beta-globin polyA, and bases 4982-5126 are the 3' AAV2 It is ITR.

[0127] AAV vectors As used herein, the term "AAV" is a general abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. Currently, there are 13 characterized serotypes of AAV, as shown in Table 1 below. Overviews and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is well known that the various serotypes are structurally and functionally very closely related, even at the genetic level, so it is quite conceivable that these same principles will be applicable to additional AAV serotypes (see, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Pattison, ed.). and Rose, Comprehensive Virology 3:1-61 (1974). For example, all AAV Serotypes, at first glance, exhibit very similar replication characteristics brought about by homologous rep genes and all have three related capsid proteins such as those expressed in AAV6. The degree of relatedness is further shown by extensive cross-hybridization between serotypes along the length of the genome; and by heteroduplex analysis that reveals the presence of similar self-annealing segments at the termini corresponding to the "inverted terminal repeats" (ITRs). Similar infectious patterns also indicate that the replication functions in each serotype are under similar regulatory control.

[0128] An "AAV vector," as used herein, refers to a vector containing one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such an AAV vector is capable of replication and packaging into infectious virus particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0129] An "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and a polynucleotide AAV vector that has formed a capsid. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene intended to be delivered to mammalian cells), it is usually referred to as an "AAV vector particle" or simply an "AAV vector." Thus, since such a vector is contained within an AAV vector particle, the production of an AAV vector particle necessarily includes the production of an AAV vector.

[0130] ​The "rep" gene and "cap" gene of AAV are genes encoding replication protein and capsid-forming protein, respectively. The rep gene and cap gene of AAV have been found in all AAV serotypes examined to date and are described in this specification and the cited references. In wild-type AAV, the rep gene and cap gene are usually found adjacent to each other within the viral genome (i.e., they are "linked" together as adjacent or overlapping transcription units), and these are usually conserved among AAV serotypes. Also, the rep gene and cap gene of AAV are individually and collectively also referred to as "AAV packaging genes". AAV in this specification The cap gene encodes a Cap protein that can package an AAV vector in the presence of rep and adeno helper functions and can bind to a target cell receptor. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a specific AAV serotype (e.g., the serotypes shown in Table 1).

Table 1

[0131] The AAV sequences used for the production of AAV may be derived from the genome of any AAV serotype. Usually, AAV serotypes have genome sequences that are significantly homologous at the amino acid level and nucleic acid level, confer a similar set of genetic functions, generate essentially physically and functionally equivalent viral particles, and replicate and construct by substantially the same mechanism. For considerations of the genome sequences and genome similarities of AAV serotypes, see, for example, GenBank accession number U89790; GenBank accession number J01901; GenBank accession number AF043303; GenBank accession number AF085716; Chlorini et al., J. Vir. 71: 6823-33 (1997); Srivastava et al., J. Vir. 45: 555-6 4 (1983); Chlorini et al., J. Vir. 73:1309-1319 (1999); Rutledge et al., J. Vi r. 72:309-319(1998); see also Wu et al., J. Vir. 74: 8635-47 (2000). .

[0132] The genomic architectures of all known AAV serotypes are very similar. The AAV genome is a linear single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique translational regions of nucleotide sequences of replication (Rep) proteins and structural (VP) proteins that are not related to the structure. The VP proteins form the capsid. The terminal 145 nt are self-complementary and are constructed such that an energetically stable intramolecular double-strand that forms a T-shaped hairpin can be formed. These hairpin structures function as the origin of viral DNA replication and serve as primers for cellular DNA polymerase complexes. The Rep gene encodes the Rep proteins, Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep52 and Rep40 are transcribed from the p19 promoter. The cap gene encodes the VP proteins, VP1, VP2, and VP3. The cap gene is transcribed from the p40 promoter.

[0133] In some embodiments, the nucleic acid sequence encoding the AAV capsid protein is operably linked to an expression control sequence for expression in a specific cell type, such as Sf9 cells or HEK cells. Methods known to those of skill in the art for expressing foreign genes in insect host cells or mammalian host cells can be used to practice the present invention. Methodologies for molecular engineering and expression of polypeptides in insect cells are described, for example, in Summers and Smith, 1986, A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow, 1991, In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors, Recombinant DNA Technology and Applications, 97-152; King, L. A. and R. D. Possee, 1992, The baculovirus expression system, Chapman and Hall, United Kingdom; O 'Reilly, D. R., L. K. Miller, V. A. Luckow, 1992, Baculovirus Expression Vecto rs: A Laboratory Manual, New York; W.H. Freeman and Richardson, C. D., 1995, B aculovirus Expression Protocols, Methods in Molecular Biology, volume 39; ​It is described in U.S. Patent No. 4,745,051; U.S. Patent Application Publication No. 2003 / 148506; and International Publication No. 03 / 074714. A promoter particularly suitable for the transcription of a nucleotide sequence encoding an AAV capsid protein is, for example, the polyhedron promoter. However, other promoters active in insect cells, such as the p10, p35, or IE-1 promoter, are known in the art, and additional promoters described in the above references are also contemplated.

[0134] The use of insect cells for the expression of heterologous proteins is well-documented in the literature, for example, methods for introducing nucleic acids, such as vectors, such as insect cell-compatible vectors, into such cells, and methods for maintaining such cells in culture media, etc. For example, METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., B ACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994) ; Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad . Sci. USA 88:4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kirnb auer et al., Vir. 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); and S amulski et al., see U.S. Patent No. 6,204,059. In some embodiments In this state, the nucleic acid construct encoding AAV in insect cells is an insect cell-compatible vector. As used herein, "insect cell-compatible vector" or "vector" refers to a nucleic acid molecule capable of the proliferative transformation or transfection of insects or insect cells. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be used as long as it is compatible with insect cells. The vector can be integrated into the insect cell genome, but the presence of the vector in the insect cell does not have to be permanent, and transient episomal vectors are also included. The vector can be introduced by any known means, for example, by chemical treatment of the cells, electroporation, or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. In a more preferred embodiment, the vector is a baculovirus, that is, the construct is a baculovirus vector. Baculovirus vectors and their methods of use are described in the above-cited references regarding the molecular engineering of insect cells.

[0135] Baculovirus is an enveloped DNA virus of arthropods, and two of its members are well-known expression vectors for producing recombinant proteins in cell culture. Baculovirus has a circular double-stranded genome (80 - 200 kbp) that can be engineered to enable the delivery of specific cell contents of the large genome. The virus used as a vector is usually Autographa californica multicapsid nucleo polyhedrovirus (AcMNPV) or Bombyx mori (Bm) NPV (Kato et al., 2010 )

[0136] Baculoviruses are commonly used for the infection of insect cells for the expression of recombinant proteins. Specifically, the expression of heterologous genes in insects can be achieved as described, for example, in U.S. Patent No. 4,745,051; Friesen et al (1986); European Patent No. 127,839; No. 155,476 ; Vlak et al (1988); Miller et al (1988); Carbonell et al (1988); Maeda et al (1985); Lebacq-Verheyden et al (1988); Smith et al (1985); Miyajima et al ( 1987); and Martin et al (1988). A number of baculovirus strains and mutants and corresponding permissive insect host cells that can be used for protein production are described in Luckow et al (1988), Miller et al (1986); Maeda et al (1985) and McKenna (1989).

[0137] Method for producing recombinant AAV ​The present disclosure provides materials and methods for producing recombinant AAV in insect cells or mammalian cells. In some embodiments, the viral construct further comprises a promoter and a restriction enzyme recognition site downstream of the promoter for allowing insertion of a polynucleotide encoding one or more target proteins, wherein the promoter and the restriction enzyme recognition site are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. In some embodiments, the viral construct further comprises a post-transcriptional regulatory element downstream of the restriction enzyme recognition site and upstream of the 3' AAV ITR. In some embodiments, the viral construct further comprises a polynucleotide inserted into the restriction enzyme recognition site and operably linked to the promoter, wherein the polynucleotide comprises a coding region of a target protein. As will be understood by those skilled in the art, any one of the AAV vectors disclosed in the present application can be used in the method as a viral construct for producing recombinant AAV.

[0138] In some embodiments, the helper function is provided by one or more helper plasmids or helper viruses comprising helper genes of adenovirus or baculovirus. Non-limiting examples of helper genes of adenovirus or baculovirus include, but are not limited to, E1A, E1B, E2A, E4, and VA, which can provide helper functions for AAV packaging.

[0139] Helper viruses of AAV are known in the art and include, for example, viruses derived from the family Adenoviridae and the family Herpesviridae. Examples of helper viruses of AAV include, but are not limited to, the SAdV-13 helper virus and SAdV-13-like helper viruses described in US Patent Application Publication No. 20110201088 (the disclosure of which is incorporated herein by reference), and the helper vector pHELP (Applied Viromics). Suitable It will be understood by those skilled in the art that any helper virus or helper plasmid of AAV that can confer helper functions to AAV can be used herein.

[0140] In some embodiments, the AAV cap gene is present within a plasmid. The plasmid further contains the AAV rep gene. Cap genes and / or rep genes derived from any AAV serotype (e.g., but not limited to, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and any variants thereof) can be used herein to generate recombinant AAV. In some embodiments, the AAV cap gene encodes a capsid derived from serotype 1, serotype 2, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13 or a variant thereof.

[0141] In some embodiments, insect cells or mammalian cells can be transfected with a helper plasmid or helper virus, a viral construct encoding the AAV cap gene, and a plasmid; and the recombinant AAV virus can be collected at various time points after co-transfection. For example, the recombinant AAV virus can be collected about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours after co-transfection, or at a time between any two of these time points.

[0142] In addition, recombinant AAV can be produced using any conventional method known in the art that is suitable for producing infectious recombinant AAV. In some examples, recombinant AAV can be produced by using insect cells or mammalian cells that stably express some of the components necessary for AAV particle production. For example, a plasmid (or plasmids) containing the AAV rep gene and the AAV cap gene, as well as a selectable marker such as the neomycin resistance gene, can be integrated into the genome of the cells. The insect cells or mammalian cells can then be co-infected with a helper virus (e.g., an adenovirus or baculovirus that provides helper functions), and a viral vector containing the 5' AAV ITR and the 3' AAV ITR (and, if desired, a nucleotide sequence encoding a heterologous protein). The advantages of this method are that the cells are selectable and are suitable for large-scale production of recombinant AAV. As another non-limiting example, instead of a plasmid, an adenovirus or baculovirus can be used to introduce the rep gene and the cap gene into the packaging cells. As yet another non-limiting example, both the viral vector containing the 5' and 3' AAV LTR and the rep-cap gene can be stably integrated into the DNA of the producer cells, and the helper function can be provided by a wild-type adenovirus to produce recombinant AAV.

[0143] Cell types used in AAV production The viral particles containing the AAV vector of the present invention can be reduced using any invertebrate cell type that enables the production of AAV or a biologic product and can be maintained in culture. For example, the insect cell lines used can be cells derived from Spodoptera frugiperda, such as SF9, SF21, SF900+, Drosophila cell lines, mosquito cell lines, such as cell lines derived from Aedes albopictus, silkworm cell lines, such as Bombyx mori cell lines, Eri silkworm cell lines, etc. It can be a Trichoplusia ni cell line, such as High Five cells or a Lepidoptera cell line, such as an Ascalapha odorata cell line. Preferred insect cells are cells derived from insect species that are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38.

[0144] Baculovirus is an enveloped DNA virus of arthropods, and two of its members are well-known expression vectors for producing recombinant proteins in cell culture. Baculovirus has a circular double-stranded genome (80 - 200 kbp) that can be engineered to enable the delivery of large genomic contents into specific cells. The virus used as a vector is generally Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or Bombyx mori (BmNPV) (Kato et al., 2010). It is (Bombyx mori) (BmNPV) (Kato et al., 2010).

[0145] Baculovirus is generally used for the infection of insect cells for the expression of recombinant proteins. Specifically, the expression of heterologous genes in insects can be achieved as described, for example, in U.S. Patent No. 4,745,051; Friesen et al (1986); European Patent No. 127,839; No. 155,4 76; Vlak et al (1988); Miller et al (1988); Carbonell et al (1988); Maeda et al (1985); Lebacq-Verheyden et al (1988); Smith et al (1985); Miyajima et al (19 87); and Martin et al (1988). The use of proteins in production A number of baculovirus strains and mutants that can be used, as well as corresponding permissive insect host cells, are described in Luckow et al (1988), Miller et al (1986); Maeda et al (1985) and M cKenna (1989).

[0146] In another aspect of the invention, the method of the invention is also carried out using any mammalian cell type that enables the replication of AAV or the production of biological products and can be maintained in culture. Preferred mammalian cells that can be used can be HEK293, HeLa, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT1080, A549, Cos-7, ARPE-19 and MRC-5 cells.

[0147] Testing of the AAV FVIII vector Assays for testing the fully packaged AAV FVIII vector of the invention include, for example, (1) in vitro checking of liver-specific mRNA expression and splicing, and the production and secretion of FVIII protein by transient introduction of a double-stranded DNA plasmid containing the AAV vector nucleic acid in HepG2 cells, a cell line derived from human liver; (2) production of AAV AAV virus particles containing the FVIII vector in 293 cells and baculovirus-infected insect cells; (3) evaluation of the AAV vector nucleic acid by alkaline gel analysis and replication assays; and (4) evaluation of FVIII expression, FVIII activity, and FVIII specific activity in Rag2 mice. These assays are described in detail in the examples.

[0148] The fully packaged AAV FVIII vector of the invention preferably exhibits at least the same expression and / or activity as the UCL SQ vector and exhibits 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more expression and / or activity compared to the UCL SQ vector.

[0149] The fully packaged AAV FVIII vector of the present invention has a high vector yield with little or no contamination of the fragmented genome, and preferably has a vector yield 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold higher than that of the UCL SQ vector.

[0150] Other aspects and advantages of the present invention will be understood by considering the following examples.

Example

[0151] Example 1 Preparation of Proto1, Proto1S, Proto2S and Proto3S vectors The UCL SQ vector is described in detail in US Patent Application Publication No. 2013 / 0024960A1 published on January 24, 2013 by Nathwani et al. (which is incorporated herein by reference in its entirety), and McIntosh et al., Blood 121: 3335-3344, 2013 and is a large-capacity AAV vector, i.e., greater than 5.0 kb. As shown in Figure 1, the UCL SQ vector contains, from left to right, AAV serotype 2 (AAV2) 5' ITR, wild-type AAV2 virus sequence, 34-base human apolipoprotein E (ApoE) / C1 enhancer, 32-base human alpha-1 antitrypsin (AAT) promoter distal X region, 186-base human AAT promoter (including a 42-base 5' untranslated region (UTR) sequence), a codon-optimized human FVIII sequence in which the B domain is replaced with a 14-amino acid SQ sequence, a 49-base synthetic polyadenylation sequence, wild-type AAV2 virus sequence, and AAV2 3' ITR. The UCL SQ vector is 5081 bases long.

[0152] To obtain a vector smaller than the UCL SQ vector, the inventors removed from the UCL SQ vector sequence DNA sequences that they considered unnecessary for FVIII expression and / or activity, or for AAV viral particle production. An exogenous DNA sequence, including 53 bases of the AAV2 viral sequence on the 3'-side of the AAV2 5' ITR, 46 bases of the AAV2 viral sequence on the 5'-side of the AAV2 3' ITR, and 11 bases adjacent to the codon-optimized FVIII SQ coding region, was removed. The resulting 4970-base-long Proto1 vector is schematically shown in FIG. 2A, and this sequence is set forth in SEQ ID NO: 1. Proto1 produces infectious virus and encodes a functional Factor VIII polypeptide.

[0153] Sequences adjacent to the hairpin loop within the AAV2 ITR may also be unnecessary in recombinant AAV vectors (Srivastava et al., U.S. Patent No. 6,521,225; Wang et al. l., J. Virol. 70:1668 - 1677, 1996; and Wang et al., J. Virol. 71:3077 - 3082, 1997 See). To further reduce the size of the Proto1 vector, 10 bases of the AAV2 sequence adjacent to the 3'-side of the hairpin loop within the AAV2 5' ITR were removed, and 10 bases of the AAV2 sequence adjacent to the 5'-side of the hairpin loop within the AAV2 3' ITR were removed. The resulting 4950-base-long Proto1S vector is schematically shown in FIG. 2B, and this sequence is set forth in SEQ ID NO: 2.

[0154] In an attempt to increase the expression of the FVIII SQ variant within the Proto1S vector, a 100-base synthetic intron was inserted between exon 1 and exon 2 within the codon-optimized FVIII sequence. It is known that intron insertion can increase the level of mRNA expression in genes that do not contain introns, such as, for example, the interferon gene.

[0155] An enhancer is defined as acting independently of distance and orientation. The 34-base ApoE / C1 enhancer acts independently of distance and orientation with respect to FVIII expression, as exemplified by its putative enhancer activity in Gray et al., U.S. Patent No. 8,030,065 (FIX expression) and Nathwani et al., U.S. Patent Application Publication No. 2013 / 0024960 (FVIII expression) (both of which are incorporated herein by reference in their entirety). The 32-base human AAT promoter distal X region described in DiSimone et al., EMBO J. 6:2759-2766, 1987 is located within a control domain that enhances the expression of heterologous promoters.

[0156] In another attempt to further increase the expression of the FVIII SQ variant in the Proto1S vector, the synthetic intron sequence incorporated the 34-base human ApoE / C1 enhancer and the 32-base human AAT promoter distal X region and was moved from its position upstream of the human AAT promoter. These two regulatory elements were inserted in an orientation opposite to their orientation in Proto1S. The resulting 4983-base Proto2S vector is schematically shown in Figure 2C and the sequence is set forth in SEQ ID NO: 3.

[0157] Since the human AAT promoter distal X region had not previously been shown to function downstream from the transcription start point within an intron, this regulatory element within the Proto2S vector was replaced with a second copy of the 34-base human ApoE / C1 enhancer in the same orientation as the first copy of the enhancer within the intron. The resulting 4985-base Proto3S vector is schematically shown in Figure 2D and this sequence is set forth in SEQ ID NO: 4.

[0158] AAV FVIII vectors in double-stranded form were generated by cloning the Proto1, Proto1S, Proto2S, and Proto3S vector nucleic acids into the pUC19 bacterial expression plasmid.

[0159] Example 2 Generation of Proto4, Proto5, Proto6 and Proto7 Vectors To further reduce the size of the Proto1 vector and / or increase the expression of FVIII compared to the Proto1 vector, the a3 domain located adjacent to the light chain or C domain was removed. The a3 domain is involved in the binding to von Willenbrand Factor, but may not be necessary for functionally active FV III in vivo.

[0160] Starting from the Proto1 vector, a 14 - amino - acid SQ sequence and a 41 - amino - acid a3 domain (corresponding to amino acids 1649 - 1689 of wild - type FVIII) were removed. The resulting Proto4 vector, 4805 bases in length, is schematically shown in Figure 3A, and this sequence is described in SEQ ID NO: 5.

[0161] In an attempt to increase the expression of B - domain - and a3 - domain - deleted FVIII, a 129 - base truncated FVIII intron was inserted between exon 1 and exon 2 in the FVIII sequence with optimized codons within the Proto4 vector. The resulting Proto5 vector, 4934 bases in length, is schematically shown in Figure 3B, and this sequence is described in SEQ ID NO: 6.

[0162] In an attempt to further increase the expression of B - domain - and a3 - domain - deleted FVIII, a second copy of the 34 - base human ApoE / C1 enhancer was inserted into the Proto5 vector in the forward or reverse orientation. The resulting Proto6 vector, 4934 bases in length and having the forward - orientation ApoE / C1 enhancer in the intron, is schematically shown in Figure 3C, and this sequence is described in SEQ ID NO: 7.

[0163] The resulting Proto7 vector, 4934 bases in length and having the reverse - orientation ApoE / C1 enhancer in the intron, is schematically shown in Figure 3D, and this sequence is described in SEQ ID NO: 8.

[0164] Double-stranded AAV FVIII vectors were generated by cloning Proto4, Proto5, Proto6, and Proto7 vector nucleic acids into the pUC19 bacterial expression plasmid.

[0165] Example 3 [[ID=**6]] Assays for Testing the Expression and Activity of AAV FVIII Vectors Assays for testing the AAV FVIII vectors of the present invention include, for example: (1) checking in vitro for liver-specific mRNA expression and splicing, and production and secretion of FVIII protein by transient introduction of a double-stranded DNA plasmid containing the AAV vector nucleic acid into HepG2 cells, a cell line derived from human liver; (2) production of AAV virus particles containing the AAV FVIII vector in 293 cells and baculovirus-infected insect cells; (3) evaluation of the AAV vector nucleic acid by alkaline gel analysis and replication assays; and (4) evaluation of FVIII expression, FVIII activity, and FVIII specific activity in Rag2 mice.

[0166] Transient Introduction Assay Preliminary in vitro assays were performed, and the expression and activity of FVIII from the AAV FVIII vectors of the present invention were compared to the expression and activity of FVIII from the UCL SQ vector. The double-stranded form of the AAV FVIII vectors of the present invention was transiently introduced into HepG2, a human hepatocyte cell line. After transfection, for example, 24 or 48 hours later, the antigen and activity of FVIII in the culture supernatant were measured.

[0167] When this assay was used, the FVIII activity in HepG2 cells transiently transfected with the Proto1, Proto1S, and Proto2S vectors was similar to the FVIII activity obtained using the UCL SQ vector, indicating that the Proto1, Proto1S, and Proto2S vectors are capable of expressing a functional factor VIII protein.

[0168] Production of AAV viral particles in 293 cells and baculovirus-infected insect cells To demonstrate that the AAV FVIII vector of the present invention indeed packages a nucleic acid encoding FVIII, the double-stranded form of the AAV FVIII vector prepared as described in Examples 1 and 2 was introduced into cells capable of producing AAV viral particles. In the first AAV viral production system, a plasmid containing the double-stranded form of the AAV FVIII vector nucleic acid was co-transfected into 293 cells with a plasmid expressing AAV Cap and Rep proteins and a plasmid expressing adenovirus helper functions necessary for AAV viral particle production. In the second AAV viral production system, a baculovirus construct expressing the AAV FVIII vector nucleic acid and a baculovirus construct expressing AAV Cap and Rep proteins were prepared and then co-infected into insect Sf9 cells. The resulting AAV viral particles produced in transiently transfected 293 cells or baculovirus-infected Sf9 cells were purified and analyzed by standard methods known in the art.

[0169] Evaluation by alkaline gel assay and replication assay An alkaline gel electrophoresis assay is used to determine the size of the packaged nucleic acid. A replication center assay is used to determine whether the AAV FVIII vector is packaged in an intact form by both packaging methods.

[0170] A primer extension assay is used to quantify the amount of AAV FVIII vector nucleic acid with intact ends, i.e., terminating at the 5' end of the hairpin loop within the AAV2 5' ITR (sense strand) or 3' ITR (antisense strand).

[0171] Alternatively, use a PCR assay to determine whether the AAV FVIII vector nucleic acid has complete ends, i.e., whether it terminates at the 5' end of the hairpin loop within the AAV2 5' ITR (sense strand) or 3' ITR (antisense strand).

[0172] Evaluation in Rag2 mice AAV viral particles produced in transiently transfected 293 cells or baculovirus-infected Sf9 cell-packaged vectors are administered intravenously to Rag2 mice at 2e11, 2e12, and 2e13 viral genomes (vg) / kg, and tested for FVIII expression and activity. Since FVIII expression and / or activity are not complicated by the presence of a host immune response to the AAV virus or human FVIII protein, Rag2 mice are used in this assay.

[0173] Determine FVIII antigen using an ELISA-based assay. Determine FVIII activity using an FXa activation assay and / or a coagulation assay. Determine the FVIII specific activity using the FVIII antigen assay and the FVIII activity assay.

[0174] By considering the presently preferred embodiments thereof, numerous variations and modifications in the practice of the invention are expected to occur to those skilled in the art. Accordingly, the only limitations to be associated with the scope of the present invention are those that appear in the appended claims.

[0175] Example 4 Preparation of constructs having improved promoter / enhancer sequences To generate additional AAV vectors with a strong promoter that increases the expression of functional FVIII, constructs with modified enhancer sequences and / or promoter sequences were generated. In some embodiments, the construct included a shortened ApoE enhancer or a μ-globulin enhancer. These constructs were generated using standard DNA cloning techniques, and their sequences are shown in SEQ ID NOs: 9-45.

[0176] Example 5 Production of AAV Viral Particles Production of Recombinant Bacmids DH10 Bac competent cells were thawed on ice. A recombinant shuttle plasmid (e.g., pFB-GFP) was added, gently mixed with the competent cells, and incubated on ice for 30 minutes. Next, the competent cells were heated at a temperature of approximately 42°C for 30 seconds and then cooled on ice for 2 minutes. The competent cells were heat shocked at 42°C for 30 seconds and cooled on ice for 2 minutes. SOC was added to the cells and incubated at 37°C for 4 hours with stirring to allow recombination to occur. During incubation, two LB plates (supplemented with various antibiotics for transformation (e.g., kanamycin, gentamicin, and tetracycline)) were coated with X-gal followed by IPTG.

[0177] An amount of the incubation mixture was obtained, diluted, and then plated onto the two LB plates and incubated at 37°C for approximately 30 - 48 hours. Several white colonies were selected from each plate and cultured overnight in LB medium containing the same combination of antibiotics provided in the LB plates. Next, bacmid DNA and glycerol stocks were prepared and stored at -80°C.

[0178] Purification of Recombinant Bacmid DNA Remove a certain amount of bacmid glycerol stock and incubate it in an LB medium containing the same combination of antibiotics as provided in the LB plates described in Example 1. Grow the culture overnight at 37 °C with shaking. Next, spin a certain amount of the said culture at maximum speed in a microcentrifuge tube for approximately 30 seconds. [[ID=,1]]

[0179] Resuspend the pellet in resuspension buffer using a pipette, then add lysis buffer, invert the tube several times to mix the buffer, and then incubate at room temperature for approximately 5 minutes. Exemplary resuspension buffer contains 50 mM Tris-Cl (pH 8.0), 10 mM EDTA, and 100 μg / mL RNaseA. Exemplary lysis buffer contains 200 mM NaOH and 1% SDS. A certain amount of precipitate buffer (for example, buffer containing 3 .0 M potassium acetate (pH 5.5)) was slowly added, the tube was inverted several times to mix the buffer, and then incubated on ice for approximately 10 minutes. Centrifuge the tube at maximum speed for approximately 10 minutes and pour the supernatant into a tube containing isopropanol. Invert the tube several times to mix the solution.

[0180] Next, centrifuge the said solution at maximum speed for approximately 15 minutes at room temperature and remove the supernatant immediately after centrifugation using a pipette.

[0181] Add a certain amount of 70% ethanol to rinse the pellet and spin again at maximum speed for 1 minute. Then remove the ethanol and spin the solution again to remove trace amounts of ethanol. Add a certain amount of TE / EB buffer to each tube and carefully dissolve the pellet with a pipette. If not used immediately, store this solution at -20 °C.

[0182] Preparation of the P0 stock of recombinant baculovirus Sf9 cells were placed in a 6-well plate at approximately 1×10 6 cells / well (or 6×10 6 cells in a 10 cm plate or 1.7×10 7Cells were seeded and allowed to attach for at least 1 hour before transfection.

[0183] Transfection solutions A and B were prepared as follows. Solution A: A certain amount of bacmid was diluted in a certain amount of serum-free medium without antibiotics in a 15 mL tube. Solution B: A certain amount of Cellfectin was diluted in a certain amount of serum-free medium without antibiotics in a 15 mL tube. Solution B was added to Solution A, gently mixed approximately 3 times with a pipette, and incubated at room temperature for 30 - 45 minutes. Next, the medium from the plate was aspirated, and a certain amount of serum-free medium without antibiotics was added to wash the cells. A certain amount of SF900II without antibiotics was added to each tube containing the lipid-DNA mixture.

[0184] The medium from the cells was aspirated, the transfection solution was added to the cells, and the cells were incubated at 28 °C for approximately 5 hours. The transfection solution was removed, a certain amount of serum-free medium and antibiotics were added, and the cells were incubated at 28 °C for approximately 4 days. The medium containing the recombinant baculovirus was collected, centrifuged at 1000 rpm for approximately 5 minutes to remove cell debris, and the baculovirus was stored at 4 °C in the dark.

[0185] Amplification of Baculovirus (P1) Sf9 cells were grown to approximately 4×10 6 cells / mL and diluted to approximately 2×10 6 cells / mL in fresh medium in a shake flask. A certain amount of Sf9 cells was infected with a certain amount of P0 stock baculovirus. The multiplicity of infection (MOI) was approximately 0.1.

[0186] The Sf9 cells were incubated for approximately 3 days and the baculovirus was harvested. The cells were spun at 2,000 rpm for 5 minutes to pellet the cells, the supernatant was collected, and stored at 4 °C in the dark. The titer of the baculovirus was determined according to the protocol of Clontech's Rapid Titer Kit.

[0187] Production of AAV Using Recombinant Baculovirus P1 Sf9 cells were grown to approximately 1×10 7 cells / mL and diluted to approximately 5×10 6 cells / mL. A certain amount of the diluted Sf9 cells was infected with Bac-vector (5 Moi) and Bac-helper (15 Moi) for 3 days. On day 3, cell viability was evaluated (approximately 50% - 70% dead cells were observed).

[0188] The cell pellet was collected by centrifugation at 3000 rpm for 10 minutes. The medium was removed and the cells were lysed (or the cell pellet was stored at -20 °C if not used immediately).

[0189] Protocol for Lysis and Banding A certain amount of Sf9 lysis buffer and Benzonase were added to the nuclear cell pellet and vortexed thoroughly to resuspend the cells. The resuspended Sf9 cells were incubated on ice for approximately 10 minutes to cool the solubilized solution. The solubilized solution was sonicated for approximately 20 seconds to fully lyse the cells and then incubated at 37 °C for approximately 30 minutes.

[0190] A certain amount of 5 M NaCl was added, the mixture was vortexed, and then incubated at 37 °C for an additional 30 minutes. A certain amount of NaCl was added to bring the salt concentration to approximately 500 mM, vortexed, and centrifuged at 8,000 rpm at 15 °C for 20 minutes to prepare a clear solubilized solution.

[0191] The clear solubilized solution was advanced to the ultracentrifugation step. A CsCl gradient was prepared by adding first the clear solubilized solution, then a certain amount of 1.32 g / cc, and then a certain amount of 1.55 g / cc CsCl solution through a syringe with a long needle. The contact surface between the CsCl solutions was marked. PBS was added to the top of the centrifuge tube, the tube was carefully balanced, and sealed.

[0192] The tubes were centrifuged at 55,000 rpm and 15 °C for approximately 20 hours. Holes were punched at the top of each tube and the AAV band located just above the contact surface mark of the two CsCl solutions was marked.

[0193] The AAV solution was transferred to centrifuge tubes for a 70.1 Ti rotor and a second CsCl centrifugation was performed, adding an amount of CsCl solution near the top of the tube. The tubes were balanced and sealed. The tubes were centrifuged at 65,000 rpm for approximately 20 hours and the AAV band (the lower band, the higher band is the empty capsid) was collected.

[0194] Example 5 Evaluation of constructs in Rag2 mice AAV genomes containing the codon-optimized SQ FVIII coding gene sequence were generated using baculovirus and 293 cells with the UCL SQ, Proto1, ProtoS1, ProtoS2, and ProtoS3 constructs. The packaging limits are 4800 bp for baculovirus and 4950 for 293 cells.

[0195] As shown in Figure 5, Proto1 with a cleaved or non-cleaved genome transduces FVIII, similar to the UCL SQ construct. 4 - 12% Bis-Tris AAV5.2 generated from baculovirus and 293T cell lysates, measured on a Gel. As shown in Figure 6, each sample expressed VP1, VP2, and VP3 proteins. As shown in Figure 7, genomic DNA obtained from the AAV samples was electrophoresed on a 0.8% alkaline agarose gel.

[0196] When these AAVs were produced by the baculovirus system, transduction of Proto1 was similar to that of the UCL SQ construct. The inclusion of intron-containing Proto2S and 3S did not transduce better than Proto1. The UCL SQ vector containing the AAV flanking sequences made in 293 cells was more potent than UCL SQ lacking the AAV sequences made within the baculovirus. As a result, in an attempt to increase the potency, additional enhancers were added to Proto1, such as constructs 101, 102, 102, and 104.

[0197] Example 6 Expression and activity of AAV FVIII vectors with improved promoter / enhancer sequences The expression and activity of AAV vectors containing constructs 99 to 106 were tested using a hydrodynamic injection protocol. Hydrodynamic delivery is a rapid method for screening liver promoters in vivo. AAV plasmid DNA was prepared using the method described in Example 5 and then diluted in TransIT-QR Hydrodynamic Delivery Solution. The plasmid DNA was injected into the tail vein of 5- to 6-week-old C57Bl / 6 mice (18-25 g) at a volume determined by (mouse weight (g) / 10) = 0.1 mL of delivery solution). The injection time was less than 5 seconds. Plasma was collected from each mouse 48 hours after injection, and the amount of expressed FVIII antigen was measured using an ELISA assay.

[0198] Increasing amounts of Proto1 plasmid (2.5, 5, 12.5, and 50 μg) were injected into the tail vein of mice. The amount of FVIII in the plasma of the injected mice was measured using an ELISA test, and recombinant FVIII (Xyntha SQ equivalent) was used as a standard for comparison.

[0199] To examine the expression, improved promoter / enhancer elements of constructs p100-400, construct 100 (p100), construct FVIII-BMN001 (pFVIII-BMN001), Proto1, construct 100AT (p100-AT), construct 100 bGH polyA (p100-bGHPA), construct 101 (p101) and construct 104 (p104). As shown in Figure 8, all constructs produced functional FVIII at various efficiency levels.

[0200] Figures 9 and 10 show data for injection of 1 μg of plasmid of various constructs. As shown in Figure 8, injection of constructs FVIII-BMN001, construct FVIII-BMN002, construct 102 (p102), construct 103 (p103) and construct 104 (p104) resulted in expression of at least 20 ng of FVIII in 5 out of 10 mice. As shown in Figure 9, injection of constructs FVIII-BMN001, construct 103 (p103), construct 103-AT (p103-AT; 398 bp hAAT promoter), construct 100 (p100), construct 100AT (p100-AT; 398 bp hAAT promoter) resulted in expression of at least 100 ng / ml of FVIII in 5 out of 10 mice.

Claims

【Claim 1】 The method described in the specification.

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