WO2015148454A1

WO2015148454A1 - Optimized and modified factor viii genes for gene therapy

Info

Publication number: WO2015148454A1
Application number: PCT/US2015/022163
Authority: WO
Inventors: Richard J. Samulski; Joshua C. GRIEGER
Original assignee: Asklepios Biopharmaceutical, Inc.
Priority date: 2014-03-28
Filing date: 2015-03-24
Publication date: 2015-10-01

Abstract

The invention relates to modified and optimized Factor VIII (FVIII) genes, nucleic acid vectors including such modified and/or optimized genes, methods of using the modified genes in the treatment of FVIII deficiencies, such as hemophilia A.

Description

OPTIMIZED AND MODIFIED FACTOR VIII GENES FOR GENE THERAPY

CROSS REFERENCE TO RELATED APPLICATION The present application and invention claims priority to U.S. Provisional Application No. 61/971,616 filed on March 28, 2014, the contents of which are incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

Field of the invention

The invention relates to modified Factor VIII (FVIII) genes, nucleic acid vectors including the modified and/or optimized genes, methods of using the modified genes in the treatment of FVIII deficiencies, such as hemophilia A.

Discussion of Related Art

Hemophilia is a blood disorder in which the blood does not clot properly. Inadequate clotting causes excessive bleeding when the hemophiliac is injured. Hemophilia is a genetic coagulation disorder characterized by inadequate clotting and excessive bleeding. Several types of hereditary hemophilia are differentiated by the clotting factor affected. Hemophilia A is caused by a deficiency in blood coagulation Factor VIII (FVIII). Gene therapy has been proposed as treatment modality for supplementing deficiencies in clotting factors in hemophiliacs. However, as in many areas of gene therapy, theory is much more straightforward than successful, effective application. Many difficulties have been encountered in prior attempts to engineer FVIII constructs that are suitable for treatment of humans.

Difficulties in implementation of gene therapy techniques include problems encountered in the use of viruses as gene vectors. While viruses are effective as gene vectors because they can be used to transduce cells leading to protein expression in vivo, the size of the factor VIII (FVIII) can be a problem.

FVIII is naturally synthesized as a single-chain polypeptide of approximately 280 kDa with the domain structure Al-al-A2-a2- -ap-A3-C l-C2 with italics denoting heavy chain acidic regions and a light chain activation peptide. Following synthesis, cleavage within the B domain results in a variably sized heavy chain (HC) (Al -al-A2-a2-B, 90- 200 kDa) and a light chain (LC) (a/?-A3-C l-C2, 80 kDa) that are associated as a heterodimer through a divalent metal ion linkage between the Al and A3 domains. Recently splitting the coding sequence into a heavy chain (HC) and light chain (LC) has been explored by using dual recombinant AAV delivery vector delivery. However, there seems to be an imbalance between the expression of HC and LC and the protein is nonfunctional.

Others have removed the B-domain of FVIII to reduce the size of the coding sequence which has been found not to compromise the biological activity of the molecule. The removal of the B-domain has been termed BDDrFVIII or recombinant B-domairi-deleted human iVIII (FVI I I-SQ). When the B domain is removed, the active form of FVIII is 170-kDA consisting of one 90-kDA and the 80-kDa light chain and wherein the entire B- domain is lacking. The smaller size of the gene provides for easier inclusion in a vector but does not provide for increased expression.

Thus, there is therefore a need for systems which overcome the shortcomings of the prior art and efficiently express the target protein in sufficient quantity to effect treatment of hemophilia A.

SUMMARY OF THE INVENTION

The present invention provides for modified Factor VIII (FVIII) genes, nucleic acid vectors including the modified and/or optimized genes, and methods of using the modified genes for increased expression levels of FVIII SQ or FVIII SQ+ and subsequent treatment of FVIII deficiencies, such as hemophilia A. In one aspect, the present invention provides for optimized FVIII genes for treating hemophilia in a human subject wherein the optimized genes have been modified to increase CG sequences and wherein nucleotides express a small number of amino acid residues of the B-domain or in the alternative all amino acid residues of the B-domain are removed. Preferably the optimized genes comprise sequences having an increased amount of CG and wherein the expressed protein (FVIII-SQ+) includes Ser 743 fused to Gin 1638 through a 14 amino acid linker from the B-domain or wherein the entire B- domain is completely removed (FVIII-SQ) and the Ser 743 residue is directly fused to the Gin 1638 residue and the B-domain is completely removed.

Preferably, the optimized nucleotide sequences encodes for the expressed protein (FVIII- SQ+)wherein the Ser 743 residue is fused to the Gin 1638 residue through a 14 amino acid linker from the B-domain and consists of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In the alternative, the optimized nucleotide sequence encodes for (FVIII-SQ) wherein the entire B-domain is removed and the Ser 743 residue is directly fused to the Gin 1638 residue and expressed by SEQ ID No: 2, SEQ ID NO: 4 or SEQ ID NO: 6.

In another aspect, the present invention provides for a virus vector comprising optimized FVIII for treating hemophilia in a human subject wherein the optimized genes have been modified to increase CG sequences and reduced cis motifs. Further the optimized genes have been modified to encode for the expressed protein (FVIII-SQ+)wherein the Ser 743 residue is fused to the Gin 1638 residue of FVIII SQ through a 14 amino acid linker from the B-domain and consisting of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In the alternative, the optimized nucleotide sequences have been modified to remove the entire B-domain (FVIII-SQ) and the Ser 743 residue is directly fused to the Gin 1638 residue as recited in SEQ ID No: 2, SEQ ID NO: 4 or SEQ ID NO: 6.

In yet another aspect, the present invention provides for a method of treating hemophilia in a subject, the method comprising:

providing at least one recombinant virus vector comprising an optimized nucleotide sequences comprising a modified FVIII gene, the optimized genes have been modified to encode for the expressed protein (FVIII- SQ+) wherein the Ser 743 residue is fused to the Gin 1638 residue through a 14 amino acid linker from the B-domain consists of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or the optimized nucleotide sequence have been modified to remove the entire B-domain (FVIII-SQ) and the Ser 743 residue is directly fused to the Gin 1638 residue consists of SEQ ID No: 2, SEQ ID NO: 4 or SEQ ID NO: 6; and

administering the recombinant virus vector to the subject under conditions such that said FVIII optimized and modified nucleotide sequences are expressed at a level which produces a therapeutically effective amount of FVIII in the subject.

Gene transfer has substantial potential use in understanding and providing therapy for disease states. There are a number of inherited diseases in which defective genes are known and have been cloned. In general, the above disease states fall into two classes: deficiency states, usually of enzymes, which are generally inherited in a recessive manner, and unbalanced states, which may involve regulatory or structural proteins, and which are typically inherited in a dominant manner. For deficiency state diseases, gene transfer could be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations. For unbalanced disease states, gene transfer could be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state. Thus the methods of the present invention permit the treatment of genetic diseases. As used herein, a disease state is treated by partially or wholly remedying the deficiency or imbalance that causes the disease or makes it more severe. In an alternative aspect, the present invention provides an expression vector comprising a polynucleotide that encodes an optimized FVIII gene. In one embodiment the expression vector is an AVV virus vector including the sequence of AAVl (SEQ ID NO: 14), AAV2 (SEQ ID NO: 12), AAV3, AAV4, AAV5, AAV6 (SEQ ID NO: 16), AAV7, AAV8, AAV 9, AAV10, AAVl l, AAV12 or chimeric variants thereof such as variant AAV2 Capsid 2.5 (SEQ ID NO. 13). Other modified sequences nucleotide sequence of modified AAV 1.1 capsid wherein amino acid residue 265 is deleted (SEQ ID NO: 15), nucleotide sequence of modified AAV 6.1 capsid wherein amino acid residue 265 is deleted (SEQ ID NO: 17), nucleotide sequence of modified AAV 6.3.1 capsid wherein amino acid residue 265 is deleted and amino acid residue 531 is changed from a Lys to a Glu (SEQ ID NO: 18). The nucleotide sequence of wildtype AAV 1 capsid is shown in (SEQ ID NO: 14) and the nucleotide sequence of wildtype AAV 6 capsid is set forth in (SEQ ID NO: 16).

In yet another aspect, the present invention provides a recombinant host cell transfected with an optimized polynucleotide that provides for increased expression of FVIII peptides. In a still further aspect, the present invention contemplates a process of preparing an increase expression of FVIII peptides, the method comprising;

transfecting a cell with an optimized polynucleotide that encodes the modified FVIII peptide selected from SEQ ID NOs: 1 to 6 to produce a transformed host cell; and

maintaining the transformed host cell under biological conditions sufficient for expression of the peptide.

In another aspect, the present invention relates to the use of an optimized FVIII gene of the present invention, that being, relative to wtFVIII, wtFVIII Refacto SQ (SEQ ID NO: 19) or wtFVIII Refacto SQ+ (SEQ ID NO: 22) in the use of a medicament for the treatment of hemophilia.

The present invention also provides for a pharmaceutical composition comprising optimized and modified FVIII genes for treating hemophilia in a human subject wherein the optimized genes have been modified to increase CG sequences and reduce cis motifs and in combination with a pharmaceutically acceptable carrier. The optimized and modified genes may comprise sequences SEQ ID NOs: 1 to 6.

Other features and advantages of the invention will be apparent from the following detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a FVIII plasmid of the present invention with all elements in an expression cassette including optimized FVIII Refacto SQ+ (SEQ ID NO: 3 with 42bp expressing 14 amino acids); TTR Promoter Truncated (SEQ ID NO: 8); MVM intron (SEQ ID NO: 9); Synthetic poly A (SEQ ID NO: 11) and collagen stability sequence (SEQ ID NO: 10).

Figure 2 shows a FVIII plasmid of the present invention with all elements in expression cassette without collagen stability sequence including FVIII Refacto SQ+ (SEQ ID NO: 3 with 42bp expressing 14 amino acids); TTR Promoter Truncated (SEQ ID NO: 8); MVM intron (SEQ ID NO: 9); and Synthetic poly A (SEQ ID NO: 11).

Figure 3 shows a FVIII plasmid of the present invention with all elements in expression cassette without MVM intron including FVIII Refacto SQ+ (SEQ ID NO: 3 with 42bp expressing 14 amino acids); TTR Promoter Truncated (SEQ ID NO: 8); Synthetic poly A (SEQ ID NO: 11) and collagen stability sequence (SEQ ID NO: 10).

Figure 4 shows a FVIII plasmid of the present invention with all elements in expression vector without MVM intron and collagen stability sequence including FVIII Refacto SQ+ (SEQ ID NO: 3 with 42bp expressing 14 amino acids); TTR Promoter Truncated (SEQ ID NO: 8); and Synthetic poly A (SEQ ID NO: 11).

Figure 5 illustrates and explains the difference between Refacto SQ+ (SEQ ID NO: 20, with 14 amino acids within SQ) and SQ (SEQ ID NO: 21 without the 14 amino acids). DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The following terms have the meanings given:

"AAV Cap" means AAV Cap proteins, VP1, VP2 and VP3 and analogs thereof.

"AAV Rep" means AAV Rep proteins and analogs thereof.

"AAV TR" means a palindromic sequence, comprising mostly complementary, symmetrically arranged sequences, and includes analogs of native AAV TRs and analogs thereof.

"Biologically-effective" with respect to an amount of a viral vector is an amount that is sufficient to result in infection (or transduction) and expression of the transgene in a target cell.

"Cis-motifs" includes conserved sequences such as found at or close to the termini of the genomic sequence and recognized for initiation of replication; cryptic promoters or sequences at internal positions likely used for transcription initiation or termination. "Chimeric" means, with respect to a viral capsid or particle, that the capsid or particle includes sequences from different parvoviruses, preferably different AAV serotypes, as described in Rabinowitz et al, U.S. Patent 6,491,907, entitled "Recombinant parvovirus vectors and method of making," granted on December 10, 2002, the disclosure of which is incorporated in its entirety herein by reference. A particularly preferred chimeric viral capsid is the AAV2.5 capsid, which has the sequence of the AAV2 capsid with the following mutations: 263 Q→A; 265 insertion T; 705 N→A; 708 V→A; and 716 T→N. wherein the nucleotide sequence expressing such capsid is defined as SEQ ID NO: 13. Additional preferred chimeric viral capsids are described in copending PCT Application No. PCT/US07/01668, the disclosure of which is incorporated in its entirety herein by reference.

"Flanked," with respect to a sequence that is flanked by other elements, indicates the presence of one or more the flanking elements upstream and/or downstream, i.e., 5' and/or 3', relative to the sequence. The term "flanked" is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and a flanking element. A sequence (e.g., a transgene) that is "flanked" by two other elements (e.g., TRs), indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be intervening sequences therebetween.

Polynucleotide" means a sequence of nucleotides connected by phosphodiester linkages. Polynucleotides are presented herein in the direction from the 5 ' to the 3 ' direction. A polynucleotide of the present invention can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule. Where a polynucleotide is a DNA molecule, that molecule can be a gene or a cDNA molecule. Nucleotide bases are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U). A polynucleotide of the present invention can be prepared using standard techniques well known to one of skill in the art.

"Transduction" of a cell by a virus means that there is transfer of DNA or RNA from the virus particle to the cell. "Transfection" of a cell means that genetic material is introduced into a cell for the purpose of genetically modifying the cell. Transfection can be accomplished by a variety of means known in the art, such as transduction or electroporation.

"Polypeptide" encompasses both peptides and proteins, unless indicated otherwise.

"Transgene" is used in a broad sense to mean any heterologous nucleotide sequence incorporated in a viral vector for expression in a target cell and associated expression control sequences, such as promoters. It is appreciated by those of skill in the art that expression control sequences will be selected based on ability to promote expression of the transgene in the target cell. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide. "Vector," means a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo.

"Recombinant" means a genetic entity distinct from that generally found in nature. As applied to a polynucleotide or gene, this means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in the production of a construct that is distinct from a polynucleotide found in nature. "Substantial homology" or "substantial similarity," means, when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the sequence. "Recombinant viral vector" means a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., polynucleotide sequence not of viral origin). In the case of recombinant parvovirus vectors, the recombinant polynucleotide is flanked by at least one, preferably two, inverted terminal repeat sequences (ITRs). "Serotype" with respect to vector or virus capsid is defined by a distinct immunological profile based on the capsid protein sequences and capsid structure.

"Peptide", "polypeptide" and "protein" are used interchangeably to denote a sequence polymer of at least two amino acids covalently linked by an amide bond.

The invention provides modified nucleic acids encoding FVIII. The invention also provides nucleic acid constructs which include as part of their sequence the modified nucleic acid encoding FVIII. For example, the invention includes plasmids and/or other vectors that include the modified FVIII = sequence along with other elements, such as regulatory elements. Further, the invention provides packaged gene delivery vehicle, such as a viral capsid, including the modified FVIII sequence. The invention also includes methods of expressing FVIII by delivering the modified sequence into a cell along with elements required to promote expression in the cell. The invention also provides gene therapy methods in which the modified FVIII sequence is administered to a subject, e.g., as a component of a vector and/or packaged as a component of a viral gene delivery vehicle. Treatment may, for example, be effected to enhance clotting in a subject and/or to treat a FVIII deficiency in the subject. Each of these aspects of the invention is discussed further in the ensuing sections.

Modified Nucleic Acid for Expression of FVIII

The invention provides a modified and optimized sequences encoding FVIII. Preferably, GC content is enhanced relative to wild-type Factor FVIII, and one or more cis-acting motifs are removed. The GC content is preferably at least 20 to 90% greater than the wild type gene. Additionally, the codon adaptation index is preferably >75, >80, >85, >90, or >95. The modified FVIII sequence may also include flanking restriction sites to facilitate subcloning into expression vector.

The invention includes a nucleic acid vector including the modified FVIII sequence and various regulatory elements. The precise nature of regulatory elements useful for gene expression will vary from organism to organism. In general, they include a promoter which directs the initiation of RNA transcription in the cell of interest. The promoter may be constitutive or regulated. Constitutive promoters are those which cause an operably linked gene to be expressed essentially at all times. Regulated promoters are those which can be activated or deactivated. Regulated promoters include inducible promoters, which are usually "off but which may be induced to turn "on," and "repressible" promoters, which are usually "on" but may be turned "off." Many different regulators are known, including temperature, hormones, cytokines, heavy metals and regulatory proteins. The distinctions are not absolute; a constitutive promoter may often be regulated to some degree. In some cases an endogenous pathway may be utilized to provide regulation of the transgene expression, e.g., using a promoter that is naturally downregulated when the pathological condition improves.

Examples of suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus promoter; the Rous Sarcoma Virus (RSV) promoter; the albumin promoter; inducible promoters, such as the Mouse Mammary Tumor Virus (MMTV) promoter; the metallothionein promoter; heat shock promoters; the a-1 -antitrypsin promoter; the hepatitis B surface antigen promoter; the transferrin promoter; the apolipoprotein A-1 promoter; human FVIII promoters; and human FIX promoters. The promoter may be a tissue-specific promoter, such as the mouse albumin promoter, which is active in liver cells as well as the transthyretin promoter (TTR).

Packaged Modified FVIII Sequence The modified FVIII sequence may also be provided as a component of a packaged viral vector or plasmid vector. The particular plasmid backbone employed in the present invention can be selected from any of the many commercially available cassettes, such as pBR322 (ATCC# 31344); pUC19 (ATCC# 37254); pcDNA3.1_ZEo (Invitrogen Cat.# V790-20), pRc/CMV (GenBank accession El 4286) obtained from Invitrogen Corporation (San Diego, CA); pXTl (GenBank accession M26398) or pSG5 (GenBank accession Af013258), obtained from Stratagene (La Jolla, CA); pPUR (GenBank accession U07648) or pMAM (GenBank accession U02443) obtained from ClonTech (Palo Alto, CA); pDual (GenBank accession # AF041247); pG51uc (GenBank accession

# AF264724); pACT (GenBank accession # AF264723); pBIND (GenBank accession # AF264722); pCI-Neo (GenBank accession # U47120); pCMV-BD (GenBank accession

# AF151088); pIRES-P (GenBank accession # Z75185); pRL-CMV (GenBank accession

# AF025843) or synthesized either denovo or by adaptation of a publicly or commercially available eukaryotic expression system. Procedures for de novo DNA synthesis are described herein below.

Viral Vector

In general, packaged viral vectors include a viral vector packaged in a capsid. Viral vectors and viral capsids are discussed in the ensuing sections.

The viral vector component of the packaged viral vectors produced according to the methods of the invention includes at least one modified FVIII sequence and associated expression control sequences for controlling expression of the modified FVIII sequence. The viral vector may include cis-acting functions sufficient to enable persistence as episomal forms or by mechanisms including integration of the modified FVIII sequence into the genome of a target cell. In a preferred embodiment, the viral vector includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and replaced by the modified hFVIII sequence and its associated expression control sequences. The modified FVIII sequence is typically inserted adjacent to one or two (i.e., flanked by) AAV TRs or TR elements adequate for viral replication; Xiao et al., J Virol 71 ;2: 941-948 (1997), in place of the viral rep and cap ORFs. Other regulatory sequences suitable for use in facilitating tissue- specific expression of the modified hFVIII sequence in the target cell may also be included.

The viral vector may be any suitable nucleic acid construct, such as a DNA or RNA construct and may be single stranded, double stranded, or duplexed.

Viral Capsid

The viral capsid component of the packaged viral vectors may be a parvovirus capsid. AAV Cap and chimeric capsids are preferred. Examples of suitable parvovirus viral capsid components are capsid components from the family Parvoviridae, such as an autonomous parvovirus or a Dependo virus. For example, the viral capsid may be an AAV capsid (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 AAV 9, AAV 10, AAV 11 or AAV 12 capsid; one skilled in the art would know there are likely other variants not yet identified that perform the same or similar function), or may include components from two or more AAV capsids. A full complement of AAV Cap proteins includes VP1, VP2, and VP3. The ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV Cap proteins may be provided.

One or more of the AAV Cap proteins may be a chimeric protein, including amino acid sequences AAV Caps from two or more viruses, preferably two or more AAVs, as described in Rabinowitz et al, U.S. Patent 6,491,907, entitled "Recombinant parvovirus vectors and method of making," granted on December 10, 2002, the entire disclosure of which is incorporated herein by reference. For example, the chimeric virus capsid can include an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. The chimeric capsid can, for example, include an AAV capsid with one or more B19 Cap subunits, e.g., an AAV Cap protein or subunit can be replaced by a B19 Cap protein or subunit. For example, in a preferred embodiment, the Vp3 subunit of the AAV capsid can be replaced by the Vp2 subunit of B19.

Production of Packaged Viral Vector

The invention includes packaging cells which may be cultured to produce packaged viral vectors of the invention. The packaging cells of the invention generally include cells with heterologous (1) viral vector function(s), (2) packaging function(s), and (3) helper function(s). Each of these component functions is discussed in the ensuing sections.

Viral Vector Functions

The packaging cells of the invention include viral vector functions, along with packaging and vector functions. The viral vector functions typically include a portion of a parvovirus genome, such as an AAV genome, with rep and cap deleted and replaced by the modified FVIII or FIX sequence and its associated expression control sequences. The viral vector functions include sufficient expression control sequences to result in replication of the viral vector for packaging. Typically, the viral vector includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and replaced by the transgene and its associated expression control sequences. The transgene is typically flanked by two AAV TRs, in place of the deleted viral rep and cap ORFs. Appropriate expression control sequences are included, such as a tissue-specific promoter and other regulatory sequences suitable for use in facilitating tissue-specific expression of the transgene in the target cell. The transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic polypeptide or a marker polypeptide. The viral vector may be any suitable nucleic acid construct, such as a DNA or RNA construct and may be single stranded, double stranded, or duplexed. The viral vector functions may suitably be provided as duplexed vector templates, as described in U.S. Patent Publication No. 2004/0029106 to Samulski et al. (the entire disclosure of which is incorporated herein by reference for its teaching regarding duplexed vectors). Duplexed vectors are dimeric self-complementary (sc) polynucleotides (typically, DNA). For example, the DNA of the duplexed vectors can be selected so as to form a double-stranded hairpin structure due to intrastrand base pairing. Both strands of the duplexed DNA vectors may be packaged within a viral capsid. The duplexed vector provides a function comparable to double-stranded DNA virus vectors and can alleviate the need of the target cell to synthesize complementary DNA to the single-stranded genome normally encapsidated by the virus.

The TPv(s) (resolvable and non-resolvable) selected for use in the viral vectors are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5 and 6 being preferred. Resolvable AAV TRs need not have a wild-type TR sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the TR mediates the desired functions, e.g., virus packaging, integration, and/or provirus rescue, and the like. The TRs may be synthetic sequences that function as AAV inverted terminal repeats, such as the "double-D sequence" as described in U.S. Pat. No. 5,478,745 to Samulski et al, the entire disclosure of which is incorporated in its entirety herein by reference. Typically, but not necessarily, the TRs are from the same parvovirus, e.g., both TR sequences are from AAV2

The packaging functions include capsid components. The capsid components are preferably from a parvoviral capsid, such as an AAV capsid or a chimeric AAV capsid function. Examples of suitable parvovirus viral capsid components are capsid components from the family Parvoviridae, such as an autonomous parvovirus or a Dependo virus. For example, the capsid components may be selected from AAV capsids, e.g., AAV1-AAV12 and other novel capsids as yet unidentified or from non human primate sources. Capsid components may include components from two or more AAV capsids.

In a more preferred embodiment, one or more of the VP capsid proteins is a chimeric protein, comprising amino acid sequences from two or more viruses, preferably two or more AAVs, as described in Rabinowitz et al, U.S. Patent 6,491,907, entitled "Recombinant parvovirus vectors and method of making," granted on December 10, 2002, the entire disclosure of which is incorporated in its entirety herein by reference. For example, the chimeric virus capsid can include a capsid region from an adeno- associated virus (AAV) and at least one capsid region from a B19 virus. The chimeric capsid can, for example, include an AAV capsid with one or more B19 capsid subunits, e.g., an AAV capsid subunit can be replaced by a B19 capsid subunit. For example, in a preferred embodiment, the VPl, VP2 or VP3 subunit of the AAV capsid can be replaced by the VPl, VP2 or VP3 subunit of B19. As another example, the chimeric capsid may include an AAV type 2 capsid in which the type 2 VPl subunit has been replaced by the VPl subunit from an AAV type 1, 3, 4, 5, or 6 capsid, preferably a type 3, 4, or 5 capsid. Alternatively, the chimeric parvovirus has an AAV type 2 capsid in which the type 2 VP2 subunit has been replaced by the VP2 subunit from an AAV type 1 , 3, 4, 5, or 6 capsid, preferably a type 3, 4, or 5 capsid. Likewise, chimeric parvoviruses in which the VP3 subunit from an AAV type 1, 3, 4, 5 or 6 (more preferably, type 3, 4 or 5) is substituted for the VP3 subunit of an AAV type 2 capsid are preferred. As a further alternative, chimeric parvoviruses in which two of the AAV type 2 subunits are replaced by the subunits from an AAV of a different serotype (e.g., AAV type 1, 3, 4, 5 or 6) are preferred. In exemplary chimeric parvoviruses according to this embodiment, the VPl and VP2, or VPl and VP3, or VP2 and VP3 subunits of an AAV type 2 capsid are replaced by the corresponding subunits of an AAV of a different serotype (e.g., AAV type 1, 3, 4, 5 or 6). Likewise, in other preferred embodiments, the chimeric parvovirus has an AAV type 1, 3, 4, 5 or 6 capsid (preferably the type 2, 3 or 5 capsid) in which one or two subunits have been replaced with those from an AAV of a different serotype, as described above for AAV type 2.

The packaged viral vector generally includes the modified FVIII sequence and expression control sequences flanked by TR elements sufficient to result in packaging of the vector DNA and subsequent expression of the modified FVIII sequence in the transduced cell. The viral vector functions may, for example, be supplied to the cell as a component of a plasmid or an amplicon. The viral vector functions may exist extrachromosomally within the cell line and/or may be integrated into the cells' chromosomal DNA.

Any method of introducing the nucleotide sequence carrying the viral vector functions into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In embodiments wherein the viral vector functions are provided by transfection using a virus vector; standard methods for producing viral infection may be used.

Packaging Functions

The packaging functions include genes for viral vector replication and packaging. Thus, for example, the packaging functions may include, as needed, functions necessary for viral gene expression, viral vector replication, rescue of the viral vector from the integrated state, viral gene expression, and packaging of the viral vector into a viral particle. The packaging functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon. The packaging functions may exist extrachromosomally within the packaging cell, but are preferably integrated into the cell's chromosomal DNA. Examples include genes encoding AAV Rep and Cap proteins.

Helper Functions The helper functions include helper virus elements needed for establishing active infection of the packaging cell, which is required to initiate packaging of the viral vector. Examples include functions derived from adenovirus, baculovirus and/or herpes virus sufficient to result in packaging of the viral vector. For example, adenovirus helper functions will typically include adenovirus components Ela, Elb, E2a, E4, and VA RNA. The packaging functions may be supplied by infection of the packaging cell with the required virus. The packaging functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon. The packaging functions may exist extrachromosomally within the packaging cell, but are preferably integrated into the cell's chromosomal DNA.

Any suitable helper virus functions may be employed. For example, where the packaging cells are insect cells, baculovirus may serve as a helper virus. Herpes virus may also be used as a helper virus in AAV packaging methods. Hybrid herpes viruses encoding the AAV Rep protein(s) may advantageously facilitate for more scalable AAV vector production schemes. Any method of introducing the nucleotide sequence carrying the helper functions into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In embodiments wherein the helper functions are provided by transfection using a virus vector or infection using a helper virus; standard methods for producing viral infection may be used.

Packaging Cell Any suitable permissive or packaging cell known in the art may be employed in the production of the packaged viral vector. Mammalian cells or insect cells are preferred. Examples of cells useful for the production of packaging cells in the practice of the invention include, for example, human cell lines, such as VERO, WI38, MRC5, A549, 293 cells, B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines.

Preferred cell lines for use as packaging cells are insect cell lines. Any insect cell which allows for replication of AAV and which can be maintained in culture can be used in accordance with the present invention. Examples include Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings concerning use of insect cells for expression of heterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al, Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al, J. Vir. 63:3822-8 (1989); Kajigaya et al, Proc. Natl Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al, J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir. 219:37- 44 (1996); Zhao et al, Vir. 272:382-93 (2000); and Samulski et al, U.S. Pat. No. 6,204,059.

During production, the packaging cells generally include one or more viral vector functions along with helper functions and packaging functions sufficient to result in replication and packaging of the viral vector. These various functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon, and they may exist extrachromosomally within the cell line or integrated into the cell's chromosomes. The cells may be supplied with any one or more of the stated functions already incorporated, e.g., a cell line with one or more vector functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, a cell line with one or more packaging functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, or a cell line with helper functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA.

Treatment Methods

The modified FVIII gene may be used for gene therapy of FVIII associated disorders, such as hemophilia A or B. An individual may be in need of gene therapy because, as a result of one or more mutations in the regulatory region and/or the coding sequence of the FVIII gene, FVIII is expressed inappropriately, e.g., has an incorrect amino acid sequence, or is expressed in the wrong tissues or at the wrong times, is underexpressed or overexpressed. The modified FVIII gene may be used as gene therapy to enhance clotting in a subject in need of enhanced clotting.

The target cells of the vectors of the instant invention are cells capable of expressing polypeptides with FVIII activity, such as those of the hepatic system of a mammal, endothelial cells and other cells with the proper cellular machinery to process the precursor to yield protein with FVIII activity. In one embodiment, the cells are normal cells cultured in vitro. The target cells may, for example, be human cells, or cells of other mammals, especially nonhuman primates and mammals of the orders Rodenta (mice, rats, rabbit and hamsters), Carnivora (cats and dogs) and Arteriodactyla (cows, pigs, sheep, goats and horses). Any cell type may be targeted. In some cases any cell type except muscle cells may be targeted.

In particular embodiments, the present invention provides a pharmaceutical composition comprising a vector of the present invention including a modified gene of FVIII in a pharmaceutically-acceptable carrier and/or other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and will preferably be in solid or liquid particulate form. As an injection medium, it is preferred to use water that contains the additives usual for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.

Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. Physiologically-acceptable carriers include pharmaceutically-acceptable carriers. Pharmaceutically acceptable carriers are those which are that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing undesirable biological effects which outweigh the advantageous biological effects of the material.

A pharmaceutical composition may be used, for example, in transfection of a cell ex vivo or in administering a viral vector or cell directly to a subject.

Recombinant virus vectors comprising the modified gene of FVIII are preferably administered to the cell in a biologically-effective amount. If the virus vector is administered to a cell in vivo (e.g., the virus is administered to a subject as described below), a biologically-effective amount of the virus vector is an amount that is sufficient to result in transduction and expression of the transgene in a target cell. The cells transduced with a viral vector are preferably administered to the subject in a "therapeutically-effective amount" in combination with a pharmaceutical carrier. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

Dosages of the cells to administer to a subject will vary upon the age, condition and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 to about 108 ,

3 8

preferably about 10 to about 10 cells, will be administered per dose. Preferably, the cells will be administered in a therapeutically-effective amount.

A further aspect of the invention is a method of treating subjects in vivo with the vector containing modified genes. Administration of the vector to a human subject or an animal in need thereof can be by any means known in the art for administering virus vectors.

Exemplary modes of administration include rectal, transmucosal, topical, transdermal, inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and the like, as well as direct tissue or organ injection, alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one may administer the virus in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

In other preferred embodiments, the inventive vector comprising the modified FVIII gene is administered intramuscularly, more preferably by intramuscular injection or by local administration. The vectors disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by administering an aerosol suspension of respirable particles comprised of the inventive parvovirus vectors, which the subject inhales. The respirable particles may be liquid or solid. Aerosols of liquid particles comprising the inventive parvovirus vectors may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Dosages of the inventive virus vector with the modified FVIII gene will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular viral vector, and the gene to be delivered, and can be determined in a routine manner. Exemplary doses for achieving therapeutic effects are virus titers of at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ transducing units or

8 13 12

more, preferably about 10 -10 transducing units, yet more preferably 10 transducing units.

The modified FVIII genes may be administered as components of a DNA molecule having regulatory elements appropriate for expression in the target cells. The modified FVIII genes may be administered as components of viral plasmids, such as rAAV vectors. Viral particles may be administered as viral particles alone, whether as an in vivo direct delivery to the portal vasculature or as an ex vivo treatment comprising administering the vector viral particles in vitro to cells from the animal receiving treatment followed by introduction of the transduced cells back into the donor.

Example 1

To assess the FVIII % activity from the Chatham plasmids, a subset of the Chatham plasmids containing wtFVIII Refacto SQ (SEQ ID NO: 19), Genscript codon-optimized FVIII Refacto SQ (SEQ ID NO 2) and GeneArt codon-optimized FVIII Refacto SQ (SEQ ID NO: 4) were hydrodynamically injected into mice. Briefly, 25 to lOOug of purified plasmid in 1-2 mL of normal saline were injected into the tail vein of mice. Delivery into the tail vein occurred within 5-10 seconds. In mice with body weight of ~20 g, injection volumes of 1.5-2.0 mL per mouse is almost equivalent to the total blood volume of the animal. The injected plasmid solution will accumulate in the inferior vena cava when the injection rate exceeds the cardiac output. A high hydrostatic pressure develops in the inferior vena cava that will force the flow of plasmid solution into tissues such as the liver, kidney and heart that are directly linked to the inferior vena cava. Since the liver is the largest organ in the body, a large portion of plasmid solution will be forced into the liver. 24 and 48 hrs post-administration, serum was collected from each mouse and assessed for FVIII protein activity (Table 1). It is apparent that the Genscript (Chatham 14 and 16 using SEQ ID NO: 2) and GeneArt (Chatham 26 and 28 using SEQ ID NO: 4) codon optimized FVIII genes in the Chatham plasmids achieved much higher FVIII protein % activity (> 10-fold) than Chatham 18 and 20 containing the wtFVIII Refacto SQ gene. No significant difference in FVIII protein activity was detected at the 48hr time point between FVIII Refacto SQ genes codon optimized by Genscript and GeneArt.

Table 1

optimized 2.0 50 100 1552 4620

Refacto SQ 2.0 50 100 995 3080

SEQ ID 2.0 50 100 2257 4074

NO: 2 2.0 50 100 728 4519

2.0 50 100 160 1096

2.0 50 100 200 918

2.0 50 100 314 1794

2.0 50 100 116 353

2.0 50 100 2367 1441

2.0 50 100 316 512

2.0 50 100 776 679

2.0 50 100 989 667

1326.1 ± 2139.0 ±

AVG ± SD

1575.8 1741.2

Chatham 26

GeneArt 2.0 50 100 2085 1269 codon 2.0 50 100 1368 1023 optimized 2.0 50 100 1899 2848

Refacto SQ 2.0 50 100 3080 435

SEQ ID 2.0 50 100 3600 5192

NO: 4

2406 ± 2153 ±

AVG ± SD

910.4 1919.1

Chatham 28

GeneArt 2.0 50 100 2534 3400 codon 2.0 50 100 3772 2386 optimized 2.0 50 100 462 1230

Refacto SQ 2.0 50 100 3485 1687

SEQ ID 2.0 50 100 890 2261

NO: 4

2229 ± 2193 ±

AVG ± SD

1497.3 818.6

Claims

The Claims

That which is claimed is: 1. An optimized and modified Factor VIII (FVIII) gene for treating hemophilia in a human subject selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6, wherein SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 encode for (FVIII-SQ+) protein and wherein SEQ ID No: 2, SEQ ID NO: 4 and SEQ ID NO: 6 encode for (FVIII-SQ).

2. The optimized and modified FVIII gene of claim 1, wherein the FVIII-SQ+ protein comprises a Ser 743 residue of SQ fused to Gin 1638 residue of SQ through a 14 amino acid linker from the B-domain and wherein the FVIII-SQ comprises a Ser 743 residue of SQ fused directly to Gin 1638 residue of SQ and all residues of the B-domain are removed.

3. The optimized gene of claim 1, where the CG sequences are increased at least 20% relative to the wild type gene.

4. The optimized gene of claim 1, included in a virus vector or plasmid vector.

5. The optimized gene of claim 4, wherein the virus vector is a chimeric virus vector comprising capsid components selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 AAV8, AAV9, AAV10, AAV11 and AAV12 capsids.

6. The optimized gene of claim 5, wherein the optimized gene is flanked by AAV TRs.

7. A method of treating hemophilia in a subject, the method comprising:

providing at least one recombinant virus vector or plasmid vector comprising a nucleotide sequences encoding an optimized and modified FVIII gene, wherein the optimized and modified FVIII gene is selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6, wherein SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 encode for (FVIII-SQ+) protein and wherein SEQ ID No: 2, SEQ ID NO: 4 and SEQ ID NO: 6 encode for (FVIII-SQ) protein; and

administering the recombinant virus vector or plasmid vector to the subject under conditions such that the optimized and modified FVIII nucleotide sequences are expressed at a level which produces a therapeutically effective amount of FVIII proteins in the subject.

8. The method of claim 7, wherein the FVIII-SQ+ protein comprises a Ser 743 residue of SQ fused to Gin 1638 residue of SQ through a 14 amino acid linker from the

B-domain and wherein the FVIII-SQ comprises a Ser 743 residue of SQ fused directly to Gin 1638 residue of SQ and all residues of the B-domain are removed.

9. The method of claim 7, where the CG sequences are increased at least 20% relative to the wild type gene.

10. The method of claim 7, wherein the recombinant virus vector is a chimeric virus vector

11. The method of claim 10, wherein the chimeric virus vector comprises capsid components selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 AAV8, AAV9, AAV10, AAV11 and AAV12 capsids.

12. A gene therapy treatment for hemophilia comprising administration of nucleotide sequence encoding for an optimized and modified FVIII gene, wherein the optimized and modified FVIII gene is selected from the group consisting of SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6, wherein SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 encode for (FVIII-SQ+) protein wherein Ser 743 residue of SQ is fused to Gin 1638 residue of SQ through a 14 amino acid linker from the B-domain, and wherein SEQ ID No: 2, SEQ ID NO: 4 and SEQ ID NO: 6 encode for (FVIII-SQ) protein and wherein Ser 743 residue of SQ is fused directly to Gin 1638 residue of SQ and all residues of the B-domain are removed.

13. The gene therapy of claim 12, wherein the FVIII-SQ+ protein comprises a Ser 743 residue of SQ fused to Gin 1638 residue of SQ through a 14 amino acid linker from the B-domain and wherein the FVIII-SQ comprises a Ser 743 residue of SQ fused directly to Gin 1638 residue of SQ and all residues of the B-domain are removed.

14. The gene therapy of claim 12, where the CG sequences are increased at least 20% relative to the wild type gene.

15. The gene therapy of claim 12, included in a virus vector or plasmid vector.

16. The gene therapy of claim 15, wherein the virus vector is a chimeric virus vector comprising capsid components selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 AAV8, AAV9, AAV10, AAV11 and AAV12 capsids.

17. A process of expressing an optimized and modified FVIII peptide comprising the steps of;

transfecting a cell with an optimized and modified FVIII gene according to claim 1 ; and maintaining the transformed host cell under biological conditions sufficient for expression of the peptide.

18. The process of claim 17, wherein the optimization is due to an increase of CG sequences of at least 20% relative to the wild type polynucleotide.

19. The process of claim 17, wherein the cell is a liver or muscle cell.

20. A pharmaceutical composition comprising an optimized and modified FVIII gene according to claim 1 ; and a pharmaceutically acceptable carrier.