HK1249117B - Optimized human clotting factor viii gene expression cassettes and their use - Google Patents

Description

Optimized human coagulation factor VIII gene expression cassette and its use

Priority Declaration

This application claims the benefit of U.S. Provisional Application Serial No. 62/112,901, filed February 6, 2015, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to synthetic liver-specific promoters and expression constructs for producing polypeptides and functional nucleic acids in the liver of a subject. The present invention also relates to Factor VIII proteins containing modifications in the amino acid sequence of the Factor VIII protein, as well as nucleic acid constructs encoding the Factor VIII proteins and methods of using these compositions to treat bleeding disorders.

Background Art

Factor VIII (FVIII) plays a key role in the coagulation cascade by accelerating the conversion of factor X to factor Xa. FVIII activity deficiency is the cause of the bleeding disorder hemophilia A. The current treatment for hemophilia A is intravenous infusion of plasma-derived or recombinant FVIII protein. Although this treatment can effectively control bleeding episodes, due to the short half-life of FVIII (8-12 hours), the requirement for frequent infusion makes it inherently costly. Gene therapy has become an attractive strategy for ultimately curing this disease. However, the progress of delivering the FVIII gene using adeno-associated virus (AAV), one of the most promising viral vectors, has lagged behind the progress of delivering coagulation factor IX because the large size of the FVIII coding sequence is close to the packaging capacity of AAV.

The present invention overcomes the shortcomings of the art by providing a short synthetic liver-specific promoter and expression construct suitable for use in AAV vectors. The present invention further provides FVIII proteins comprising additional glycosylation sites with amino acid sequence modifications and methods for use thereof in treating bleeding disorders.

Summary of the Invention

The present invention is based in part on the development of a synthetic liver-specific promoter that is only about 200 base pairs long. This promoter can be used to produce polypeptides and functional nucleic acids in a liver-specific manner, particularly using AAV vectors, which have strict length restrictions and can benefit from the availability of short but strong promoters.

The present invention is also based in part on the development of modified FVIII proteins comprising additional glycosylation sites in the heavy chain. The modified proteins provide prolonged and high levels of activity relative to FVIII proteins without the modifications described herein.

In one aspect, the present invention relates to a polynucleotide comprising a synthetic liver-specific promoter, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

In another aspect, the present invention relates to vectors, cells and/or transgenic animals comprising a polynucleotide of the present invention.

In another aspect, the present invention relates to a method for producing a polypeptide or functional nucleic acid in a subject's liver, comprising delivering the polynucleotide, vector and/or transformed cell of the present invention to a subject, thereby producing the polypeptide or functional nucleic acid in the subject's liver.

In an additional aspect, the invention relates to a method of treating hemophilia A in a subject, comprising delivering to the subject a therapeutically effective amount of a polynucleotide, vector and/or transformed cell of the invention, thereby treating hemophilia A in the subject.

In another aspect, the present invention relates to a method of increasing the bioavailability of a Factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide, vector and/or transformed cell of the present invention, thereby increasing the bioavailability of the Factor VIII polypeptide in the subject.

In yet another aspect, the present invention relates to a modified human Factor VIII polypeptide, wherein amino acid residues in the heavy chain are modified to create one or more glycosylation sites.

In additional aspects, the present invention relates to a polynucleotide encoding a modified human Factor VIII polypeptide of the present invention and vectors, cells and/or transgenic animals comprising the polynucleotide.

In another aspect, the present invention relates to a method for producing Factor VIII in the liver of a subject, comprising delivering to the subject a polynucleotide encoding a modified human Factor VIII polypeptide of the present invention, or a vector and/or transformed cells comprising the polynucleotide, thereby producing Factor VIII in the liver of the subject.

In another aspect, the present invention relates to a method for treating hemophilia A in a subject, comprising delivering to the subject a therapeutically effective amount of a modified human Factor VIII polypeptide, polynucleotide, vector and/or transformed cell of the present invention, thereby treating hemophilia A in the subject.

In an additional aspect, the present invention relates to a method for increasing the bioavailability of a Factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide encoding a modified human Factor VIII polypeptide of the present invention, or a vector and/or transformed cells comprising the polynucleotide, thereby increasing the bioavailability of the Factor VIII polypeptide in the subject.

These and other aspects of the invention are set forth in more detail in the description of the invention which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the sequence of the LXP3.3 promoter (SEQ ID NO: 1). Putative liver and housekeeping transcription factor binding sites are highlighted by underlining.

Figure 2A shows a comparison of LacZ expression in the liver and heart after intravenous injection of AAV9-TBG-LacZ or AAV9-Lxp3.3-LacZ into mice. Shown are X-gal and H&E double staining of thin sections of liver and heart.

FIG2B shows a quantitative comparison of LacZ enzyme activity in various tissues of mice treated with 1×10 ¹¹ vector genomes (vg) of AAV9-LacZ vectors containing a nonspecific CMV promoter, a liver-specific TBG promoter, or a LXP3.3 promoter (as shown in FIG1 ).

FIG2C shows a quantitative comparison of LacZ enzyme activity in the livers of mice treated with the AAV9-LacZ vectors as in FIG2B but at a lower dose (2×10 ¹⁰ vector genomes (vg)).

Figure 3 shows Factor VIII activity in supernatants of Huh7 cells transfected with different AAV vector plasmids containing the NBP promoter (177 bp) driving the human BDD Factor VIII gene without introns or containing a VH4 intron or a chimeric CIN intron.

Figure 4 shows the sequence of the AAV-Lxp3.3i-BDD-F8 construct and the Lxp3.3i promoter-intron (SEQ ID NO: 2). ITR stands for the 145 bp inverted terminal repeat sequence of AAV.

FIG5 shows FVIII expression in human hepatoma Huh7 cells and mouse Hepa1-6 cells using two constructs containing either the nonspecific CMV promoter or the liver-specific LXP3.3 promoter.

FIG6 shows FVIII activity in Huh7 cells transfected with different BDD Factor VIII (synthetic opti-F8 or wild-type wtF8) plasmids containing the promoter LXP3.3 or the weak promoter TkPro, respectively.

Figure 7A shows long-term human FVIII gene expression and FVIII activity mediated by AAV9-Lxp3.3-F8 in FVIII knockout mice following IV injection of a high dose (2 x 10 ¹¹ vg/mouse) or a low dose (4 x 10 ¹⁰ vg/mouse). Factor VIII activity (as a percentage of normal human levels) was measured 2, 6, 10, and 18 weeks after injection.

Figure 7B shows long-term human FVIII gene expression and human FVIII protein concentration mediated by AAV9-Lxp3.3-F8 in FVIII knockout mice after IV injection of high dose (2×10 ¹¹ vg/mouse) or low dose (4×10 ¹⁰ vg/mouse). Factor VIII protein (as a percentage of normal human levels) was measured by ELISA at 2, 6, 10, and 18 weeks after injection.

Figure 8 shows the amino acid sequences of modified BDD FVIII proteins (SEQ ID NOs: 31-34). SQ represents Serine 743 (Ser 743) and Glutamine (Gln 1638) at the junction of the heavy and light chains of BDD Factor VIII (numbering based on SEQ ID NO: 5). Underlined letters highlight the mutated amino acids in the heavy chain.

FIG9A shows human FVIII activity in Huh7 cells transfected with expression plasmids containing BDD Factor VIII or mutants F8X1 and F8X2 (see FIG8 for details).

Figure 9B shows AAV8 vector-mediated long-term expression of the mutant human BDD FVIII gene in FVIII knockout mice. Human factor VIII activity (as a percentage of normal human levels) was measured at various time points after intravenous injection of ^5x1010 vector genomes of AAV8-LXP3.3i-F8X1 or AAV8-LXP3.3i-F8X2.

DETAILED DESCRIPTION

The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. However, the present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination. Furthermore, the invention also contemplates that, in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. For illustration, if the specification states that a compound comprises components A, B, and C, it is specifically intended that any one or combination of A, B, or C may be omitted or disregarded, alone or in any combination.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terms used in the description of the invention herein are only for the purpose of describing particular embodiments and are not intended to limit the invention.

Unless otherwise expressly indicated, nucleotide sequences are presented herein only as a single strand, from left to right in the 5' to 3' direction. Nucleotides and amino acids are referred to herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids) by the single-letter code or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

Unless otherwise indicated, standard methods known to those skilled in the art can be used for cloning genes, amplifying and detecting nucleic acids, etc. These techniques are known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY, 1989); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

definition

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.

Furthermore, as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted as an alternative ("or").

As used herein, the term "about" when referring to a measurable value, such as an amount of a polypeptide, dosage, time, temperature, enzyme activity or other biological activity, etc., is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified amount.

The transitional phrase “consisting essentially of” means that the scope of a claim should be construed to include the specified materials or steps recited in the claim “and such other matters which do not materially affect the basic and novel characteristics of the claimed invention.” See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis original); see also MPEP §2111.03.

The term "consisting essentially of..." (and grammatical variations) as applied to a polynucleotide or polypeptide sequence of the present invention refers to a polynucleotide or polypeptide consisting of the sequence (e.g., SEQ ID NO) and a total of ten or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids at the 5' and/or 3' or N-terminus and/or C-terminus of the sequence, such that the function of the polynucleotide or polypeptide is not substantially altered. The total of ten or fewer additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added at both ends. The term "substantially altered" as applied to a polynucleotide of the present invention means that the ability to express the encoded polypeptide is increased or decreased by at least about 50% or more compared to the expression level of a polynucleotide consisting of the sequence. The term "substantially altered" as applied to a polypeptide of the present invention means that the coagulation stimulating activity is increased or decreased by at least about 50% or more compared to the activity of a polypeptide consisting of the sequence.

As used herein, the terms "enhance" or "increase" or grammatical variations thereof, refer to an increase in a specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold or even 15-fold.

As used herein, the term "inhibit" or "reduce" or grammatical variations thereof refers to a decrease or reduction in a specified level or activity by at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In specific embodiments, the inhibition or reduction results in little or essentially no detectable activity (at most an insignificant amount, e.g., less than about 10% or even 5%).

As used herein, an "effective" amount is an amount that provides the desired effect.

As used herein, a "therapeutically effective" amount is an amount that provides some improvement or benefit to a subject. Alternatively, a "therapeutically effective" amount is an amount that will provide some relief, alleviation, or reduction in at least one clinical symptom in a subject. One skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

As used herein, a "prophylactically effective" amount is an amount sufficient to prevent (as defined herein) a disease, disorder and/or clinical symptoms in a patient. Those skilled in the art will understand that the level of prevention need not be complete as long as some benefit is provided to the subject.

As is well known to those skilled in the art, the efficacy of treating a bleeding disorder by the methods of the present invention can be determined by detecting clinical improvement as indicated by changes in the subject's symptoms and/or clinical parameters.

The terms "treat," "treating," or "treatment" are intended to alleviate or at least partially improve or alter the severity of a subject's condition and to achieve some relief, alleviation, or reduction in at least one clinical symptom.

The terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to a reduction or delay in the extent or severity of a disease, disorder, and/or clinical symptom after onset relative to what would have occurred had the methods of the invention not been performed prior to the onset of the disease, disorder, and/or clinical symptom. In the context of hemophilia A, "prevention" refers to a reduction in the number and/or severity of bleeding episodes compared to the number and/or severity of bleeding episodes that would have occurred in the absence of prophylactic treatment.

As used herein, "nucleic acid," "nucleotide sequence," and "polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The terms polynucleotide, nucleotide sequence, or nucleic acid refer to a chain of nucleotides regardless of chain length. Nucleic acids can be double-stranded or single-stranded. When single-stranded, the nucleic acid can be a sense strand or an antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids with altered base pairing ability or increased nuclease resistance. The present invention also provides a nucleic acid that is a complement (which can be a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of the present invention.

An "isolated polynucleotide" is a nucleotide sequence (e.g., DNA or RNA) that is not directly adjacent to the nucleotide sequences to which it is directly adjacent (one at the 5' end and one at the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequence immediately adjacent to the coding sequence. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic organism, or that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment generated by PCR or restriction endonuclease treatment) that is not associated with other sequences. It also includes recombinant DNA that is part of a hybrid nucleic acid that encodes an additional polypeptide or peptide sequence. An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such a gene, but rather includes the coding region and regulatory regions associated with the gene, but without the additional genes naturally occurring on that chromosome.

The term "fragment" as applied to polynucleotides will be understood to refer to a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence, and comprising, consisting essentially of, and/or consisting of a nucleotide sequence of consecutive nucleotides that are identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments according to the present invention may be included in larger polynucleotides of which they are a component, where appropriate. In some embodiments, such fragments may comprise, consist essentially of, and/or consist of an oligonucleotide having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the present invention.

The term "isolated" can refer to a nucleic acid, nucleotide sequence, or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Additionally, an "isolated fragment" is a fragment of a nucleic acid, nucleotide sequence, or polypeptide that is not a naturally occurring fragment and would not be found in nature. "Isolated" does not mean that the preparation is technically pure (homogeneous), but that it is sufficiently pure to provide the polypeptide or nucleic acid in a form useful for its intended purpose.

The term "fragment" as applied to polypeptides will be understood to refer to an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence, and comprising, consisting essentially of, and/or consisting of an amino acid sequence of consecutive amino acids that are identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such polypeptide fragments according to the present invention may be included in larger polypeptides of which they are a component, where appropriate. In some embodiments, such fragments may comprise, consist essentially of, and/or consist of a peptide having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive amino acids of a polypeptide or amino acid sequence according to the present invention.

"Vector" is any nucleic acid molecule for nucleic acid cloning and/or transfer to a cell. A vector can be a replicon to which another nucleotide sequence can be attached to allow the attached nucleotide sequence to replicate. A "replicon" can be any genetic element (such as a plasmid, phage, cosmid, chromosome, viral genome) as an autonomous unit (i.e., capable of replicating under its own control) of nucleic acid replication in vivo. The term "vector" includes viruses and non-viral (such as plasmid) nucleic acid molecules that are introduced into cells in vitro, in vitro, and/or in vivo. Nucleic acid can be manipulated using a large number of vectors known in the art, response elements and promoters can be merged into genes, etc. For example, the nucleic acid fragment corresponding to a response element and a promoter can be inserted into a suitable vector, which can be achieved by connecting a suitable nucleic acid fragment to a selected vector with complementary binding ends. Alternatively, the end of the nucleic acid molecule can be enzymatically modified, or any site can be produced by connecting a nucleotide sequence (joint) to the nucleic acid end. Such a vector can be engineered to contain a sequence encoding a selective marker, which facilitates the selection of cells containing the vector and/or the nucleic acid of the vector to be merged into the cell genome. Such markers allow identification and/or selection of host cells that incorporate and express the protein encoded by the marker.A "recombinant" vector refers to a viral or non-viral vector comprising one or more heterologous nucleotide sequences (i.e., a transgene), for example comprising two, three, four, five or more heterologous nucleotide sequences.

Viral vectors have been used in various gene delivery applications in cells and living animal subjects. Operable viral vectors include but are not limited to retrovirus, slow virus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus and adenovirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectin (cytofectin)), nucleic acid-protein complexes and biopolymers. Except nucleic acid of interest, carrier can also include one or more regulatory regions and/or can be used for selecting, measuring and monitoring nucleic acid transfer results (delivery to specific tissues, expression duration etc.) selectable markers.

Vectors can be introduced into the desired cells by methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or a nucleic acid carrier transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990). In various embodiments, other molecules can be used to facilitate delivery of nucleic acids in vivo, such as cationic oligopeptides (e.g., WO 95/21931), peptides derived from nucleic acid binding proteins (e.g., WO 96/25508), and/or cationic polymers (e.g., WO 95/21931). Vectors can also be introduced in vivo as naked nucleic acids (see U.S. Pat. Nos. 5,693,622, 5,589,466, and 5,580,859). Receptor-mediated nucleic acid delivery methods can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).

As used herein, the terms "protein" and "polypeptide" are used interchangeably and include peptides and proteins unless otherwise indicated.

A "fusion protein" is a polypeptide produced when two heterologous nucleotide sequences encoding two (or more) different polypeptides, or fragments thereof, that are not found fused together in nature, are fused together in the correct translation reading frame. Illustrative fusion polypeptides include fusions of a polypeptide of the present invention (or fragment thereof) with all or part of glutathione-S-transferase, maltose binding protein, or a reporter protein (e.g., green fluorescent protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, a FLAG epitope, or the like.

As used herein, a "functional" polypeptide or "functional fragment" is a substance that substantially retains at least one biological activity normally associated with the polypeptide (e.g., angiogenic activity, protein binding, ligand or receptor binding). In specific embodiments, a "functional" polypeptide or "functional fragment" retains substantially all activities possessed by the unmodified peptide. By "substantially retaining" biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 99% or more of the biological activity of the native polypeptide (and may even have a higher level of activity than the native polypeptide). A "non-functional" polypeptide is a polypeptide that exhibits little or substantially no detectable biological activity normally associated with the polypeptide (e.g., at most an insignificant amount, such as less than about 10% or even 5%). Biological activities such as protein binding and angiogenic activity can be measured using assays well known in the art and as described herein.

The term "express" or "expressed" a polynucleotide coding sequence means that the sequence is transcribed and optionally translated. Generally, according to the present invention, expression of a coding sequence of the present invention will result in the production of a polypeptide of the present invention. The expressed polypeptide or fragment can also function in intact cells without purification.

In the context of the present invention, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV, as well as any other AAV now known or later discovered. See, for example, BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers). Several additional AAV serotypes and clades have been identified (see, for example, Gao et al., (2004) J. Virol. 78: 6381-6388 and Table 1), which are also encompassed by the term "AAV."

The genomic sequences of various AAVs and autonomous parvoviruses, as well as the sequences of ITRs, Rep proteins, and capsid subunits are known in the art. These sequences can be found in the literature or in public databases, such as databases. See, for example, accession numbers NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC 001358, NC001540, AF513851, AF513852, AY530579, AY631965, AY631966; the disclosures of which are incorporated herein in their entirety. See also, e.g., Srivistava et al., (1983) J. Virol. 45:555; Chiorini et al., (1998) J. Virol. 71:6823; Chiorini et al., (1999) J. Virol. 73:1309; Bantel-Schaal et al., (1999) J. Virol. 73:939; Xiao et al., (1999) J. Virol. 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; International Patent Publication Nos. WO 00/28061, WO 99/61601, WO 98/11244; U.S. Patent No. 6,156,303; the disclosures of which are incorporated herein in their entireties. See also Table 1. Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, PA (incorporated herein in its entirety) provides an early description of the terminal repeat sequences of AAV1, AAV2, and AAV3.

Table 1

A "recombinant AAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) comprising at least one inverted terminal repeat (e.g., one, two, or three inverted terminal repeat sequences) and one or more heterologous nucleotide sequences. rAAV vectors typically retain the 145 base terminal repeats (TRs) in cis to produce the virus; however, modified AAV TRs and non-AAV TRs (including partially or completely synthetic sequences) can also be used for this purpose. All other viral sequences are unnecessary and can be provided in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158: 97). The rAAV vector optionally comprises two TRs (e.g., AAV TRs), which will typically be at the 5' and 3' ends of the heterologous nucleotide sequence, but not necessarily adjacent thereto. The TRs can be identical to or different from each other. The vector genome can also contain a single ITR at its 3' or 5' end.

The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates a desired function such as replication, viral packaging, integration, and/or proviral rescue, etc.). The TR can be an AAV TR or a non-AAV TR. For example, non-AAV TR sequences, such as those of other parvoviruses (e.g., canine parvovirus (CPV), parvovirus of mice (MVM), human parvovirus B-19), or the sequence of the SV40 hairpin that serves as the SV40 origin of replication, can be used as TRs, which can be further modified by truncations, substitutions, deletions, insertions, and/or additions. In addition, the TR can be partially or completely synthetic, such as the "double D sequence" described in U.S. Pat. No. 5,478,745 to Samulski et al.

"AAV terminal repeats" or "AAV TRs" can be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any other AAV now known or later discovered (see, e.g., Table 1). The AAV terminal repeats need not have native terminal repeat sequences (e.g., native AAV TR sequences can be altered by insertions, deletions, truncations, and/or missense mutations), as long as the terminal repeats mediate the desired function, e.g., replication, viral packaging, integration, and/or proviral rescue, etc.

The terms "rAAV particle" and "rAAV virion" are used interchangeably herein. An "rAAV particle" or "rAAV virion" comprises an rAAV vector genome packaged within an AAV capsid.

AAV capsid structure is described in more detail in BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

The term "pharmacokinetic properties" has its usual and customary meaning and refers to the absorption, distribution, metabolism, and excretion of the FVIII protein.

The usual and customary meaning of "bioavailability" is the fraction or amount of an administered dose of a bioactive drug that reaches the systemic circulation. In the context of embodiments of the present invention, the term "bioavailability" includes the usual and customary meaning, but is also considered to have a broader meaning to include the degree of bioactivity of the FVIII protein. In the case of FVIII, for example, one measure of "bioavailability" is the procoagulant activity of the FVIII protein available in the circulation after infusion.

"Post-translational modification" has the usual and customary meaning and includes, but is not limited to, removal of leader sequences, gamma-carboxylation of glutamic acid residues, beta-hydroxylation of aspartic acid residues, N-linked glycosylation of asparagine residues, O-linked glycosylation of serine and/or threonine residues, sulfation of tyrosine residues, phosphorylation of serine residues, and any combination thereof.

As used herein, "biological activity" is determined by reference to a standard, for example, from human plasma. For FVIII, the standard may be TRANSACTION® (CSL Behring). The biological activity of the standard is taken as 100%.

The term "factor VIII protein" or "FVIII protein" as used herein includes wild-type FVIII protein and naturally occurring or artificial proteins (e.g., B domain deleted proteins). The FVIII proteins of the present invention may further include mutant forms of FVIII known in the literature. The FVIII proteins of the present invention also include any other naturally occurring human FVIII proteins or artificial human FVIII proteins now known or later identified, as well as their derivatives and active fragments/active domains known in the art.

The amino acid sequences of FVIII from various mammalian species are available from sequence databases such as GenBank. Examples of FVIII sequences are shown in the table below.

species GenBank accession number Humans (Homo sapiens) AAA52484.1 House mouse (Mus musculus) NP_032003.2 Wild boar (Sus scrofa) AAB06705.1 Bos Taurus NP_001138980.1 Domestic dog (Canis lupus familiaris) NP-001003212.1 Brown rat (Rattus norvegicus) ADU79112.1

The FVIII proteins of the present invention also include pharmacologically active forms of FVIII, which are molecules from which the signal peptide has been removed and the B domain has been removed by the action of a protease (or engineered from the protein by removing it at the nucleic acid level), resulting in two discontinuous polypeptide chains (light chain and heavy chain) of FVIII that fold into a functional FVIII coagulation factor. Several B domain-deleted forms of human FVIII are known, including the frequently used SQ version, in which the residues between S743 and Q1638 are deleted. In particular, modified FVIII proteins with increased glycosylation are specifically encompassed in a broad sense.

The amino acid sequence of human FVIII protein is well known in the art and can be found in GenBank Accession No. AAA52484. Human FVIII protein is 2351 amino acids in length and consists of a signal peptide (residues 1-19), a heavy chain (residues 20-759), a B domain (residues 760-1332), and a light chain (residues 1668-2351). The amino acid sequence without the signal peptide is disclosed below (SEQ ID NO: 5).

The term "half-life" is a broad term that includes the usual and customary meanings and the usual and customary meanings found in the FVIII scientific literature. Specifically included in this definition are measurements of parameters related to FVIII, which define the time after infusion from the initial value measured at the time of infusion to the time half of the initial value is reduced. In some embodiments, antibodies to FVIII can be used in various immunoassays to measure the half-life of FVIII in blood and/or blood components, as known in the art and as described herein. Alternatively, functional assays including standard coagulation assays can be used to measure half-life as a reduction in FVIII activity, as known in the art and as described herein.

The term "recovery" as used herein includes, after its infusion, injection, delivery or other mode administration, the amount of FVIII measured by any acceptable method in a recipient animal or human subject (e.g., in the circulation) at the earliest practical time of taking out a biological sample (e.g., blood or blood product sample) in order to measure the level of FVIII, including but not limited to the FVIII antigen level or the FVIII protease or coagulation activity level detected. Using existing methods, the earliest biological sampling time for measuring FVIII recovery is usually within the first 15 minutes after the infusion, injection or other mode delivery/administration of FVIII, but with the improvement of science and/or clinical technology, it is reasonable to expect faster sampling times. In essence, the recovery value of FVIII is represented here, at the earliest possible time point after infusion, injection or other mode delivery to a recipient animal or patient, the maximum fraction of FVIII that can be measured in a recipient (e.g., in the circulation) by infusion, injection or other mode delivery/administration.

The term "glycosylation site" is a broad term with its usual and customary meaning. In the context of this application, the term applies to both sites that can potentially accept carbohydrate moieties as well as sites within a protein, particularly FVIII, to which carbohydrate moieties have actually been attached and includes any amino acid sequence that can serve as an acceptor for oligosaccharides and/or carbohydrates.

As used herein, a "transformed" cell is a cell that has been transformed, transduced, and/or transfected with a nucleic acid molecule encoding a FVIII protein of the present invention, including but not limited to a FVIII protein vector constructed using recombinant DNA technology.

As used herein, the term "bleeding disorder" reflects any defect of cellular, physiological, or molecular origin, congenital, acquired, or induced, that manifests as bleeding. Examples are coagulation factor deficiencies (e.g., hemophilia A and B or deficiencies of coagulation factors XI, VII, VIII, or IX), coagulation factor inhibitors, platelet dysfunction, thrombocytopenia, von Willebrand's disease, or bleeding caused by surgery or trauma.

Excessive bleeding also occurs in subjects with a normally functioning blood coagulation cascade (without coagulation factor deficiency or inhibitors for any coagulation factor) and may be caused by platelet insufficiency, thrombocytopenia, or von Willebrand disease. In this case, bleeding may be similar to that caused by hemophilia because the hemostatic system (as in hemophilia) lacks or has abnormal essential coagulation "compounds" (such as platelets or von Willebrand factor proteins), leading to major bleeding. In subjects experiencing extensive tissue damage associated with surgery or trauma, normal hemostatic mechanisms may be overwhelmed by the need for immediate hemostasis, so bleeding may still occur despite normal hemostatic mechanisms. When bleeding in organs such as the brain, inner ear region, and eyes, the possibility of surgical hemostasis is limited, and achieving satisfactory hemostasis is also a problem. The same problem may also occur during biopsy in various organs (liver, lung, tumor tissue, gastrointestinal tract) and laparoscopic surgery. What all of these situations have in common is that it is difficult to provide hemostasis by surgical techniques (sutures, clips, etc.), also when the bleeding is diffuse (hemorrhagic gastritis and heavy uterine bleeding). Acute and heavy bleeding may also occur in subjects on anticoagulant therapy, in which defective hemostasis is induced by the treatment given to the subject. In the case where the anticoagulant effect must be quickly offset, these subjects may require surgical intervention. Radical retropubic prostatectomy is a conventional surgery for subjects with localized prostate cancer. The surgery is often complicated by significant and sometimes massive blood loss. The considerable blood loss during prostatectomy is mainly related to the complex anatomical situation, which has various densely vascularized sites where surgical hemostasis is not easy to obtain, and may lead to large areas of diffuse bleeding. In addition, intracerebral hemorrhage is the most untreatable form of stroke and is associated with high mortality and hematoma growth in the first few hours after intracerebral hemorrhage. Another situation that may cause problems in the case of poor hemostasis is when a subject with normal hemostatic mechanisms is treated with anticoagulant therapy to prevent thromboembolic disease. Such treatments may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists, and aspirin and other platelet aggregation inhibitors.

In one embodiment of the invention, the bleeding is associated with hemophilia. In another embodiment, the bleeding is associated with hemophilia with acquired inhibitors. In another embodiment, the bleeding is associated with thrombocytopenia. In another embodiment, the bleeding is associated with von Willebrand's disease. In another embodiment, the bleeding is associated with severe tissue damage. In another embodiment, the bleeding is associated with severe trauma. In another embodiment, the bleeding is associated with surgery. In another embodiment, the bleeding is associated with laparoscopic surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis. In another embodiment, the bleeding is heavy uterine bleeding. In another embodiment, the bleeding occurs in an organ with limited possibility of mechanical hemostasis. In another embodiment, the bleeding occurs in the brain, inner ear region, or eyes. In another embodiment, the bleeding is associated with the process of taking a biopsy. In another embodiment, the bleeding is associated with anticoagulant therapy.

"Subjects" of the present invention include any animal suffering from or susceptible to a bleeding disorder or bleeding condition that requires and/or desires to control bleeding, which can be treated, improved, or prevented by administering FVIII to the subject (e.g., hemophilia A and acquired FVIII deficiency (e.g., due to autoantibodies to FVIII or hematological malignancies). Such subjects are typically mammalian subjects (e.g., laboratory animals such as rats, mice, guinea pigs, rabbits, primates, etc.), farm or commercial animals (e.g., cows, horses, goats, donkeys, sheep, etc.), or livestock (e.g., cats, dogs, ferrets, etc.). In specific embodiments, the subject is a primate subject, a non-human primate subject (e.g., chimpanzee, baboon, monkey, gorilla, etc.), or a human. The subject of the present invention can be a subject known or believed to have a bleeding disorder or bleeding condition risk that requires and/or desires to be controlled. Alternatively, the subject according to the present invention may also include a subject previously unknown or suspected of having a bleeding disorder or bleeding condition risk that requires or desires to be controlled. As another alternative, the subject can be a laboratory animal and/or an animal model of the disease.

The subject includes males and/or females of any age, including neonates, infancy, adulthood and elderly subjects. With regard to human subjects, in representative embodiments, the subject can be an infant (e.g., less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months or less age), a toddler (e.g., at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months) or a child (e.g., at least about 1, 2, 3, 4 or 5 years old and/or less than about 14 years old, 12, 10, 8, 7, 6, 5 or 4 years old). In embodiments of the invention, the subject is a human subject of about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 12 to 18, 24, 36, 48 or 60 months of age, a human subject of about 18 to 24, 30, 36, 48 or 60 months of age, or a human subject of about 24 to 30, 36, 48 or 60 months of age.

Promoter and expression cassette

One aspect of the present invention relates to a polynucleotide comprising a synthetic liver-specific promoter, wherein the promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 or a sequence having at least about 90% identity thereto. In some embodiments, the nucleotide sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO: 1. The promoter is a short (approximately 200 base pairs) and strong liver-specific promoter that is ideal for liver-specific expression of the polynucleotide of interest and is particularly suitable for use in AAV vectors due to its short length and the limited capacity of AAV vectors. The promoter is designed to contain a conserved basic promoter element and a transcription start site. The basic promoter is linked to a number of liver-specific transcription factor binding sites at its 5' end for liver-specific expression (Figure 1). The promoter was initially identified in vitro using a luciferase reporter gene and transfection experiments in the human hepatoma cell line Huh7, and then confirmed in vivo in mice to exhibit high activity.

In some embodiments, the promoter is part of an expression cassette in which it is operably linked to an intron, for example, at the 3' end of the promoter. This can be done to increase the expression level of the polynucleotide of interest linked to the promoter. Any suitable intron can be used, for example, the chimeric intron CIN (Promega). In some embodiments, the intron is from VH4. In some embodiments, the intron may further comprise a short non-natural exon junction sequence. In one embodiment, the promoter and intron together comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 2, or a sequence having at least 90% identity thereto (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO: 1).

The promoter may be operably linked to a polynucleotide of interest. In some embodiments, the polynucleotide of interest encodes a polypeptide or a functional nucleic acid. In certain embodiments, the polynucleotide of interest encodes a coagulation factor, such as FVIII, such as a FVIII with a B domain deletion. The FVIII with a B domain deletion may be encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 90% identity thereto (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto), or consisting essentially of, or consisting of, the nucleotide sequence of SEQ ID NO: 3. In one embodiment, the expression cassette comprising a promoter, an intron, and a polynucleotide of interest encoding a B-domain deleted FVIII comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 4, or a sequence having at least 90% identity thereto (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO: 4).

Another aspect of the present invention is a vector comprising a polynucleotide of the present invention, such as an expression vector. The vector can be any type of vector known in the art, including but not limited to plasmid vectors and viral vectors. In some embodiments, the viral vector is a retroviral or lentiviral vector. In some embodiments, the viral vector is an AAV vector from any known AAV serotype, including but not limited to AAV type 1, AAV type 2, AAV type 3 (including type 3A and type 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV11, bird AAV, cattle AAV, dog AAV, horse AAV and sheep AAV, as well as any other AAV now known or later discovered. In some embodiments, the AAV vector is AAV8 or AAV9.

Another aspect of the present invention relates to cells (e.g., isolated cells, transformed cells, recombinant cells, etc.) comprising a polynucleotide and/or vector of the present invention. Thus, various embodiments of the present invention relate to recombinant host cells containing a vector (e.g., an expression cassette). Such cells can be isolated and/or present in a transgenic animal. Transformation of cells is further described below.

Another aspect of the present invention relates to transgenic animals comprising the polynucleotides, vectors and/or transformed cells of the present invention. Transgenic animals are further described below.

The polynucleotides, vectors and/or cells of the invention may be included in pharmaceutical compositions. Some embodiments relate to kits comprising the polynucleotides, vectors and/or cells of the invention, and/or reagents and/or instructions for use of the kits, e.g., to practice the methods of the invention.

Modified Factor VIII protein

One aspect of the present invention relates to modified mammalian factor VIII polypeptides (e.g., human FVIII polypeptides), wherein the amino acid residues in the heavy chain are modified to produce one or more additional glycosylation sites. In certain embodiments, the one or more additional glycosylation sites are located in the C-terminus of the heavy chain, such as the last 100 amino acid residues of the heavy chain, such as the last 50, 40, 30, 20, 10, 9, 8, 7, 6, or 5 residues. In some embodiments, the polypeptide is modified to produce at least 2 glycosylation sites, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more glycosylation sites. Modifications can include amino acid substitutions, additions, deletions, or any combination thereof. These modifications are introduced into the amino acid sequence of the FVIII protein to produce a FVIII protein with increased activity after expression in vivo.

"Additional" glycosylation sites refers to the number of glycosylation sites in the FVIII protein being greater than the number of glycosylation sites typically present in an unmodified (eg, wild-type) FVIII protein (eg, SEQ ID NO: 5).

The present invention also relates to FVIII proteins containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) additional sugar side chains. Such additional sugar side chains can be present at one or more glycosylation sites in the FVIII proteins of the present invention. Alternatively, as is well known to those skilled in the art, the additional sugar side chains can be present at sites on the FVIII protein as a result of chemical and/or enzymatic methods for introducing such sugar chains into the FVIII molecule. "Additional" or "new" sugar chains refers to the number of sugar chains in the FVIII protein that is greater than the number of sugar chains normally present in the "wild-type" form of FVIII. In various embodiments, from about 1 to about 50 additional sugar side chains can be added (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

The glycosylation site can be an N-linked glycosylation site, an O-linked glycosylation site, or a combination of an N-linked glycosylation site and an O-linked glycosylation site. In some embodiments, the added glycosylation site comprises an N-linked glycosylation site, and the consensus sequence is NXT/S, with the proviso that X is not proline. In other embodiments, the glycosylation site comprises an O-linked glycosylation site comprising a consensus sequence selected from the group consisting of CXXGGT/SC (SEQ ID NO: 24), NSTE /DA (SEQ ID NO: 25), NI T QS (SEQ ID NO: 26), QSTQS (SEQ ID NO: 27), D/EF T- R/KV (SEQ ID NO: 28), CE/D- SN (SEQ ID NO: 29), GG S C-K/R (SEQ ID NO: 30), and any combination thereof.

In some embodiments, from about 1 to about 15 glycosylation sites can be added to the amino acid sequence of a FVIII protein of the invention. In various embodiments, from about 1 to about 50 glycosylation sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) can be added.

As used herein, "glycosylation attachment site" or "glycosylation site" can refer to a sugar attachment consensus sequence (i.e., a series of amino acids that serves as a consensus sequence for attaching a sugar (monosaccharide, oligosaccharide, or polysaccharide) to an amino acid sequence) or it can refer to the actual amino acid residue to which the sugar moiety is covalently linked. The sugar moiety can be a monosaccharide (simple sugar molecule), an oligosaccharide, or a polysaccharide.

In a specific embodiment, additional amino acids can be inserted between any amino acid residues that make up the heavy chain and/or substituted with such amino acid residues. In addition, the same insert of the present invention can be introduced multiple times at the same and/or different positions in the amino acid sequence of the FVIII protein. In addition, different inserts and/or the same insert can be introduced one or more times at the same and/or different positions between amino acid residues throughout the amino acid sequence of the FVIII protein.

Some proteins can support a large number of sugar side chains, and the distance between N-linked glycosylation sites can be as few as three, four, five, or six amino acids (see, e.g., Lundin et al., FEBS Lett. 581:5601 (2007); Apweiler et al., Biochim. Biophys. Acta 1473:4 (1991), the entire contents of which are incorporated herein by reference).

In some embodiments, amino acid residues 736 and 737 of the wild-type human sequence (SEQ ID NO: 5) are replaced with amino acid residue XX, wherein X is S or T. Thus, residues 736 and 737 can be SS, ST, TS, or TT.

In some embodiments, amino acid residues 736-742 of the wild-type human sequence (SEQ ID NO: 5) are replaced with amino acid residues XXYVNRXL (SEQ ID NO: 6), wherein X is S or T. Thus, residues 736-742 may be as follows.

Residues 736-742 SEQ ID NO TTYVNRSL 7 TTYVNRTL 8 TSYVNRSL 9 TSYVNRTL 10 STYVNRSL 11 STYVNRTL 12 SSYVNRSL 13 SSYVNRTL 14

In some embodiments, amino acid residues 736-742 in the wild-type human sequence (SEQ ID NO: 5) are replaced with amino acid residues XXNNX (SEQ ID NO: 15), wherein X is S or T. Thus, residues 736-740 may be as follows.

In some embodiments, the modified human Factor VIII polypeptide is one in which the B domain is deleted, such as an SQ deletion from S743 to Gln1638 (as numbered in SEQ ID NO: 5).

The FVIII proteins of the present invention having additional glycosylation sites can be produced by recombinant methods such as site-directed mutagenesis using PCR. Alternatively, the FVIII proteins of the present invention can be chemically synthesized to prepare FVIII proteins having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) additional glycosylation sites.

It is within the skill of those skilled in the art and within the scope of the present invention to modify any amino acid residue or residues in the mature FVIII amino acid sequence according to methods well known in the art and as taught herein, and to test any resulting FVIII protein for activity, stability, recovery, half-life, etc. according to methods well known in the art and as taught herein (see, e.g., Elliott et al., J. Biol. Chem. 279: 16854 (2004), the entire contents of which are incorporated herein by reference).

Embodiments of the present invention relate to recombinant FVIII proteins (e.g., X0, X1, X2) to which glycosylation sites have been added to improve the activity and/or recovery and/or half-life and/or stability of FVIII. The FVIII proteins of the present invention comprise modifications that allow for improved bioavailability of the FVIII protein to a subject to which the FVIII protein of the present invention has been administered. In some embodiments, improved bioavailability refers to standard current thinking in hematology, where the concentration of FVIII in plasma is the relevant concentration. In some embodiments of the present invention, improved bioavailability refers to the ability of the FVIII protein to remain in the circulation of a subject for a longer period of time. Therefore, in some embodiments of the present invention, the FVIII proteins described herein are modified to produce FVIII proteins with increased activity after in vivo expression, and in some embodiments, the present invention provides a method for increasing the hemostatic effectiveness of a FVIII protein in a subject, comprising administering to the subject an effective amount of a FVIII protein of the present invention, a polynucleotide of the present invention, a vector of the present invention, and/or a cell of the present invention, wherein the FVIII protein administered to the subject in any of these embodiments is a FVIII protein of the present invention with increased activity.

The FVIII proteins according to the present invention are produced and characterized by methods well known in the art and described herein. As is well known in the art, these methods include measuring clotting time (partial thromboplastin time (PPT) assay) and administering the FVIII protein to test animals to determine recovery, half-life, and bioavailability by appropriate immunoassays and/or activity assays.

Another aspect of the present invention provides an isolated polynucleotide encoding the FVIII protein of the present invention and an expression cassette for producing the FVIII protein.

Another aspect of the present invention is a vector comprising a polynucleotide of the present invention, such as an expression vector. The vector can be any type of vector known in the art, including but not limited to plasmid vectors and viral vectors. In some embodiments, the viral vector is an AAV vector from any known AAV serotype, including but not limited to AAV type 1, AAV type 2, AAV type 3 (including type 3A and type 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, bird AAV, bovine AAV, dog AAV, horse AAV and sheep AAV, as well as any other AAV now known or later discovered. In some embodiments, the AAV vector is AAV8 or AAV9.

Another aspect of the present invention relates to cells (e.g., isolated cells, transformed cells, recombinant cells, etc.) comprising a polynucleotide and/or vector of the present invention. Thus, various embodiments of the present invention relate to recombinant host cells containing the vector (e.g., expression cassette). Such cells can be isolated and/or present in a transgenic animal. Transformation of cells is further described below.

Another aspect of the present invention relates to transgenic animals comprising the polynucleotides, vectors and/or transformed cells of the present invention. Transgenic animals are further described below.

The FVIII proteins, polynucleotides, vectors and/or cells of the invention can be included in pharmaceutical compositions. Some embodiments relate to kits comprising the FVIII proteins, polynucleotides, vectors and/or cells of the invention, and/or reagents and/or instructions for using the kits, e.g., to practice the methods of the invention.

Method of the present invention

Another aspect of the present invention relates to promoter of the present invention and expression cassette and is used for producing the purposes of polypeptide or functional nucleic acid in a liver-specific manner, for example.Therefore, one aspect relates to the method for producing polypeptide or functional nucleic acid in the liver of experimenter, comprises and sends polynucleotide of the present invention, carrier and/or transformed cell to experimenter, thereby produces polypeptide or functional nucleic acid acid in the liver of experimenter.Described polynucleotide, carrier and/or transformed cell are sent under the condition that interested polynucleotide expression occurs, to produce polypeptide or functional nucleic acid.These conditions are well-known in the art and are described further below.

Another aspect of the present invention relates to a method of treating hemophilia A or acquired factor VIII deficiency in a subject using the promoter and expression cassette of the present invention, comprising delivering to the subject a therapeutically effective amount of a polynucleotide, vector and/or transformed cell of the present invention, thereby treating hemophilia A in the subject. In some embodiments, the polynucleotide of interest encodes a FVIII polypeptide as described above.

Another aspect of the present invention relates to a method for increasing the bioavailability of a FVIII polypeptide in a subject using the promoter and expression cassette of the present invention, comprising delivering to the subject an effective amount of a polynucleotide, vector and/or transformed cell of the present invention, thereby increasing the bioavailability of the FVIII polypeptide in the subject. In this aspect, the polynucleotide of interest encodes a FVIII polypeptide as described above.

The modified FVIII proteins of the present invention can be used in methods for treating bleeding disorders by administering an effective amount of the FVIII protein to a subject in need thereof (e.g., a human patient). Accordingly, the present invention also provides methods for treating bleeding disorders, comprising administering an effective amount of the FVIII protein, polynucleotide, vector, and/or cell of the present invention to a subject in need thereof.

One aspect of the present invention relates to a method of producing Factor VIII in the liver of a subject, comprising delivering to the subject a polynucleotide encoding a modified human Factor VIII polypeptide, a vector and/or a transformed cell of the present invention, thereby producing Factor VIII in the liver of the subject.

Another aspect of the invention relates to a method of treating hemophilia A or acquired factor VIII deficiency in a subject, comprising delivering to the subject a therapeutically effective amount of a modified human factor VIII polypeptide, polynucleotide, vector and/or transformed cell of the invention, thereby treating hemophilia A or acquired factor VIII deficiency in the subject.

Another aspect of the present invention relates to a method for increasing the bioavailability of a Factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide encoding a modified human Factor VIII polypeptide, a vector and/or a transformed cell of the present invention, thereby increasing the bioavailability of the Factor VIII polypeptide in the subject.

Bleeding disorders that can be treated according to the methods of the present invention include any disorder that can be treated with FVIII, such as hemophilia A and acquired FVIII deficiency. Such treatment protocols and dosing regimens for administering or delivering a FVIII protein of the present invention and/or a polynucleotide encoding a FVIII protein of the present invention to a subject (e.g., a subject in need thereof) are well known in the art.

In an embodiment of the present invention, the dosage of a vector encoding a FVIII protein of the present invention (e.g., a viral vector or other nucleic acid vector) can be an amount that achieves a therapeutic plasma concentration of the FVIII protein. The therapeutic concentration of the FVIII protein is considered to be a normal level above 1% of healthy individuals, which is measured on an average of 100%, thus being one international unit (IU) of FVIII in 1 mL of normal human plasma. One skilled in the art will be able to determine the optimal dosage for a given subject and a given condition.

For treatment associated with intentional intervention, the FVIII protein of the invention is typically administered within about 24 hours prior to the intervention and for up to 7 days or more thereafter.Administration as a coagulant can be by a variety of routes as described herein.

The pharmaceutical composition is primarily used for parenteral administration for preventive and/or therapeutic treatment. Preferably, the pharmaceutical composition is administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly, or by continuous or pulse infusion. Alternatively, the pharmaceutical composition can be formulated for administration in various ways, including but not limited to oral, subcutaneous, intravenous, intracerebral, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, intraocular, or any other acceptable manner.

Compositions for parenteral administration comprise the FVIII protein of the present invention in combination with (e.g., dissolved in) a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, and the like. Compositions that extend stability and storage (e.g., methionine and sucrose) can also be used to formulate the FVIII protein of the present invention. The FVIII protein of the present invention can also be formulated into liposome formulations for delivery or targeting to sites of injury. Liposomal formulations are generally described in U.S. Patent Nos. 4,837,028, 4,501,728, and 4,975,282. The composition can be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solution can be packaged for use, or filtered and lyophilized under aseptic conditions, and the lyophilized formulation can be combined with a sterile aqueous solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjustment and buffering agents, tension regulators, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like. The composition may further contain a preservative, an isotonic agent, a nonionic surfactant or detergent, an antioxidant and/or other various additives.

The concentration of FVIII protein in these formulations can vary widely, i.e., from less than about 0.5% by weight (typically at least about 1% by weight) to as much as about 15% or 20% by weight, and will be selected primarily based on the specific mode of administration selected, by fluid volume, viscosity, etc. Thus, as a non-limiting example, a typical pharmaceutical composition for intravenous infusion can be prepared to contain 250 ml of sterile Ringer's solution and 10 mg of FVIII protein. Actual methods for preparing parenterally administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences , 21st edition, Mack Publishing Company, Easton, Pa. (2005).

Compositions comprising a FVIII protein of the present invention and/or a nucleic acid molecule encoding a FVIII protein of the present invention can be administered for prophylactic and/or therapeutic treatment. In therapeutic applications, the composition is administered to a subject already suffering from the disease as described above in an amount sufficient to cure, alleviate, or partially prevent the disease and its complications. An amount sufficient to achieve this goal is defined as a "therapeutically effective amount." As will be appreciated by those skilled in the art, the amount effective for this purpose will depend on the severity of the disease or injury, as well as the subject's weight and general condition.

In prophylactic applications, a composition containing a FVIII polypeptide of the invention is administered to a subject susceptible to or otherwise at risk of a disease state or injury to enhance the subject's own coagulation ability. Such an amount is defined as a "prophylactically effective dose." In prophylactic applications, the precise amount will again depend on the subject's health and weight.

Single or multiple administrations of the composition can be carried out at a dose level and pattern selected by the treating physician.For ambulatory subjects requiring daily maintenance levels, the FVIII protein can be administered by continuous infusion using, for example, an ambulatory pump system.

The FVIII proteins of the present invention can also be formulated as sustained or extended release formulations. Methods for formulating sustained or extended release compositions are known in the art and include, but are not limited to, semipermeable matrices of solid hydrophobic particles containing the polypeptide.

Local delivery of the FVIII protein of the present invention, such as topical administration, can be performed, for example, by spraying, perfusion, double-balloon catheters, stents, incorporation into vascular grafts or stents, hydrogels for coating balloon catheters, or other established methods. In any case, the pharmaceutical composition should provide an amount of the FVIII protein sufficient to effectively treat the subject.

In some embodiments, an AAV vector is used to deliver a polynucleotide of interest (e.g., a FVIII protein) to a subject. Accordingly, the present invention also provides an AAV viral particle (i.e., a virion) comprising a polynucleotide of interest, wherein the viral particle packages (i.e., encapsidates) a vector genome, optionally an AAV vector genome.

In a specific embodiment, a virion is a recombinant vector comprising a heterologous polynucleotide of interest (e.g., for delivery to a cell). Thus, the present invention can be used to deliver polynucleotides to a cell in vitro, in vitro, and in vivo. In representative embodiments, the recombinant vector of the present invention can be advantageously used to deliver or transfer polynucleotides into animal (e.g., mammalian) cells.

Any heterologous nucleotide sequence can be delivered by the viral vectors of the present invention.Polynucleotides of interest include those encoding polypeptides, optionally therapeutic (eg, for medical or veterinary use) and/or immunogenic (eg, vaccine) polypeptides.

Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator (CFTR), dystrophin (including protein products of dystrophin mini-genes or micro-genes, see, e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Application No. 2003017131; Wang et al., (2000) Proc. Natl. Acad. Sci. USA 97:13714-9 [min-dystrophin]; Harper et al., (2002) Nature Genetics 5:13714-1371 ... Med. 8: 253-61 [micro-dystrophin]); mini-agrin, laminin-α2, sarcoglycan (α, β, γ, or δ), Fukutin-related protein, myostatin propeptide, follistatin, dominant negative myostatin, angiogenic factors (e.g., VEGF, angiopoietin-1 or 2), anti-apoptotic factors (e.g., heme oxygenase-1, TGF-β, inhibitors of pro-apoptotic signaling such as caspases, proteases, kinases, death receptors [e.g., CD-095], cytochrome c release regulators, inhibitors of mitochondrial pore opening and swelling); activin type II soluble receptor, anti-inflammatory polypeptides such as Ikappa B dominant mutant, sarcospan, utrophin, mini-utrophin, antibodies or antibody fragments directed against myostatin or myostatin propeptide, cell cycle regulators, Rho kinase regulators such as Cethrin, which is a modified bacterial C3 exoenzyme [available from BioAxone Therapeutics, Inc., Saint-Lauren, Quebec, Canada], BCL-xL, BCL2, XIAP, FLICEc-s, dominant negative caspase-8, dominant negative caspase-9, SPI-6 (see, e.g., U.S. Patent Application No. 20070026076), transcription factor PGC-o1, Pinch gene, ILK gene and thymosin 4 gene), coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, intracellular and/or extracellular superoxide dismutase, leptin, LDL receptor, neprilysin, lipoprotein lipase, ornithine transcarbamylase, β-globulin, α-globulin, spectrin, α ₁ -antitrypsin, methylcytosine binding protein 2, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., interferon-α, interferon-β, interferon-γ, interleukins-1 to -14, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., growth hormone (somatotropin), insulin, insulin-like growth factors including IGF-1 and IGF-2, GLP-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factors-3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor-α and -β, etc.), bone morphogenetic protein ( including RANKL and VEGF), lysosomal proteins, glutamate receptors, lymphokines, soluble CD4, Fc receptors, T cell receptors, ApoE, ApoC, inhibitor of protein phosphatase inhibitor 1 (I-1), phospholamban, serca2a, lysosomal acid α-glucosidase, α-galactosidase A, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), calsarcin, receptors (e.g., soluble tumor necrosis growth factor-α receptor), anti-inflammatory factors such as IRAP, Pim-1, PGC-1α, SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-β4, hypoxia-inducible transcription factor [HIF], angiogenic factors, S 100A1, parvalbumin, adenylyl cyclase type 6, molecules that affect G protein-coupled receptor kinase type 2 knockout (such as truncated constitutively active bARKct); phospholamban inhibitory or dominant-negative molecules such as phospholamban S16E, monoclonal antibodies (including single-chain monoclonal antibodies) or suicide gene products (such as thymidine kinase, cytosine deaminase, diphtheria toxin and tumor necrosis factor such as TNF-α), and any other polypeptides that have a therapeutic effect in a subject in need thereof.

The heterologous nucleotide sequence of coded polypeptide includes those of coding reporter polypeptide (such as enzyme).Reporter polypeptide is known in the art, includes but is not limited to fluorescent protein (such as EGFP, GFP, RFP, BFP, YFP or dsRED2), produces the enzyme of detectable product, such as luciferase (such as from Gaussia, Renilla or Photinus), β-galactosidase, β-glucuronidase, alkaline phosphatase, and chloramphenicol acetyltransferase gene, or can directly detect protein.Almost any protein can be directly detected by using the specific antibody of such as the protein.Sambrook and Russell (2001), Molecular Cloning, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al. (1992), Current Protocols in Molecular Biology, John Wiley＆Sons (including regular updates) disclose other markers (and related antibiotics) applicable to the positive or negative selection of eukaryotic cells.

Alternatively, the heterologous nucleic acid can encode a functional RNA, such as an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that affects spliceosome-mediated trans-splicing (see Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), an interfering RNA (RNAi), including small interfering RNA (siRNA) that mediates gene silencing (see Sharp et al., (2000) Science 287:2431), a microRNA or other non-translated "functional" RNA, such as a "guide" RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), and the like. Exemplary non-translated RNAs include RNAi or antisense RNAs to multidrug resistance (MDR) gene products (e.g., for treating tumors and/or administering to the heart to prevent damage from chemotherapy), RNAi or antisense RNAs to myostatin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNAs to VEGF or tumor immunogens (including but not limited to those specifically described herein) (for treating tumors), RNAi or antisense oligonucleotides to mutant dystrophin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNAs to the hepatitis B surface antigen gene (to prevent and/or treat hepatitis B infection), RNAi or antisense RNAs to the HIV tat and/or rev genes (to prevent and/or treat HIV), and/or RNAi or antisense RNAs to any other immunogen from a pathogen (to protect a subject from a pathogen) or a defective gene product (to prevent or treat disease). RNAi or antisense RNAs to the above targets or any other targets can also be used as research reagents.

As is known in the art, antisense nucleic acids (e.g., DNA or RNA) and inhibitory RNA (e.g., microRNA and RNAi such as siRNA or shRNA) sequences can be used to induce "exon skipping" in patients with muscular dystrophy resulting from a defect in the dystrophin gene. Thus, the heterologous nucleic acid can encode an antisense nucleic acid or inhibitory RNA that induces appropriate exon skipping. It will be understood by those skilled in the art that the specific method of exon skipping depends on the nature of the underlying defect in the dystrophin gene, and many such strategies are known in the art. Exemplary antisense nucleic acid and inhibitory RNA sequences target upstream branch points and/or downstream donor splice sites and/or internal splicing enhancer sequences of one or more dystrophin exons (e.g., exon 19 or 23). For example, in specific embodiments, the heterologous nucleic acid encodes an antisense nucleic acid or inhibitory RNA directed against the upstream branch point and downstream splice donor site of exon 19 or 23 of the dystrophin gene. Such sequences can be incorporated into AAV vectors that deliver modified U7 snRNA and antisense or inhibitory RNA (see, e.g., Goyenvalle et al., (2004) Science 306: 1796-1799). As another strategy, modified U1 snRNA can be incorporated into AAV vectors along with siRNA, microRNA, or antisense RNA complementary to upstream and downstream splice sites of dystrophin exons (e.g., exon 19 or 23) (see, e.g., Denti et al., (2006) Proc. Nat. Acad. Sci. USA 103: 3758-3763). In addition, antisense and inhibitory RNAs can target splicing enhancer sequences within exons 19, 43, 45, or 53 (see, e.g., U.S. Pat. No. 6,653,467; U.S. Pat. No. 6,727,355; and U.S. Pat. No. 6,653,466).

Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes possess a specific catalytic domain that possesses endonuclease activity (Kim et al., (1987) Proc. Natl. Acad. Sci. USA 84:8788; Gerlach et al., (1987) Nature 328:802; Forster and Symons, (1987) Cell 49:211). For example, a number of ribozymes accelerate phosphoester transfer reactions with high specificity, typically cleaving only one of the multiple phosphoesters in an oligonucleotide substrate (Michel and Westhof, (1990) J. Mol. Biol. 216:585; Reinhold-Hurek and Shub, (1992) Nature 357:173). This specificity has been attributed to the requirement that the substrate bind to the ribozyme's internal guide sequence ("IGS") through specific base-pairing interactions prior to the chemical reaction.

Ribozyme catalysis has been observed primarily as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, (1989) Nature 338:217). For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with sequence specificity greater than that of known ribozymes and approaching that of DNA restriction enzymes. Therefore, sequence-specific ribozyme-mediated inhibition of nucleic acid expression may be particularly useful for therapeutic applications (Scanlon et al., (1991) Proc. Natl. Acad. Sci. USA 88:10591; Sarver et al., (1990) Science 247:1222; Sioud et al., (1992) J. Mol. Biol. 223:831).

MicroRNA (mir) is a natural cellular RNA molecule that can regulate the expression of a variety of genes by controlling the stability of mRNA. Overexpression or reduction of specific microRNAs can be used to treat functional disorders and has been shown to be effective in many disease states and animal models of diseases (see, for example, Couzin, (2008) Science 319: 1782-4). Chimeric AAV can be used to deliver microRNA to cells, tissues, and subjects for the treatment of genetic and acquired diseases, or for enhancing the functionality and growth promotion of certain tissues. For example, mir-1, mir-133, mir-206, and/or mir-208 can be used to treat heart and skeletal muscle diseases (see, for example, Chen et al., (2006) Genet. 38: 228-33; van Rooij et al., (2008) Trends Genet. 24: 159-66). MicroRNA can also be used to regulate the immune system after gene delivery (Brown et al., (2007) Blood 110: 4144-52).

As used herein, the term "antisense oligonucleotide" (including "antisense RNA") refers to a nucleic acid that is complementary to and specifically hybridizes to a specified DNA or RNA sequence. Antisense oligonucleotides and nucleic acids encoding the antisense oligonucleotides can be prepared according to conventional techniques. See, for example, U.S. Patent No. 5,023,243 to Tullis; U.S. Patent No. 5,149,797 to Pederson et al.

Those skilled in the art will understand that the antisense oligonucleotide need not be completely complementary to the target sequence, so long as the degree of sequence similarity is sufficient to enable the antisense nucleotide sequence to specifically hybridize to its target (as defined above) and reduce the production of a protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be performed under reduced stringency, moderate stringency or even stringent conditions. Suitable conditions for achieving reduced, moderate and stringent hybridization conditions are as described herein.

Alternatively, in specific embodiments, the antisense oligonucleotides of the invention have at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or more sequence identity to the complement of the target sequence and reduce the production of a protein product (as defined above). In some embodiments, the antisense sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches compared to the target sequence.

Methods for determining percent nucleic acid sequence identity are described in more detail elsewhere herein.

The length of the antisense oligonucleotide is not critical, as long as it specifically hybridizes to the predetermined target and reduces the production of a protein product (as defined above) and can be determined according to routine procedures. Typically, the length of the antisense oligonucleotide is at least about 8, 10, or 12 or 15 nucleotides and/or less than about 20, 30, 40, 50, 60, 70, 80, 100, or 150 nucleotides.

RNA interference (RNAi) is another useful method for reducing the production of protein products (e.g., shRNA or siRNA). RNAi is a mechanism of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) corresponding to a target sequence of interest is introduced into a cell or organism, resulting in degradation of the corresponding mRNA. The mechanisms by which RNAi achieves gene silencing are reviewed in Sharp et al., (2001) Genes Dev 15:485-490; and Hammond et al., (2001) Nature Rev. Gen. 2:110-119). The RNAi effect persists for multiple cell divisions before gene expression is regained. Therefore, RNAi is a powerful method for targeted knockout or "knockdown" at the RNA level. RNAi has been shown to be successful in human cells, including human embryonic kidney and HeLa cells (see, for example, Elbashir et al., Nature (2001) 411:494-8).

Initial attempts to use RNAi in mammalian cells generated an antiviral defense mechanism involving PKR in response to dsRNA molecules (see, e.g., Gil et al., (2000) Apoptosis 5:107). Short synthetic dsRNAs of approximately 21 nucleotides, termed "short interfering RNAs" (siRNAs), have been shown to mediate silencing in mammalian cells without eliciting an antiviral response (see, e.g., Elbashir et al., Nature (2001) 411:494-8; Caplen et al., (2001) Proc. Nat. Acad. Sci. USA 98:9742).

RNAi molecules (including siRNA molecules) can be short hairpin RNAs (shRNAs; see Paddison et al., (2002), Proc. Nat. Acad. Sci. USA 99: 1443-1448), which are believed to be processed into 20-25-mer siRNA molecules in cells by the action of the RNase III-like enzyme Dicer. shRNAs typically have a stem-loop structure in which two inverted repeat sequences are separated by a short spacer sequence that loops out. shRNAs with loops of 3 to 23 nucleotides in length have been reported. The loop sequence is generally not important. Exemplary loop sequences include the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA.

RNAi can further comprise a circular molecule comprising a sense and antisense region, with two loop regions on either side thereof so as to form a "dumbbell" shaped structure when dsRNA is formed between the sense and antisense regions. The molecule can be treated in vitro or in vivo to release the dsRNA portion, such as siRNA.

International Patent Publication No. WO 01/77350 describes a vector for bidirectional transcription to produce sense and antisense transcripts of a heterologous sequence in eukaryotic cells. This technology can be used to generate RNAi for use in accordance with the present invention.

Shinagawa et al., (2003) Genes Dev. 17: 1340 reported a method for expressing long dsRNA from a CMV promoter (pol II promoter), which is also applicable to tissue-specific pol II promoters. Similarly, the method of Xia et al., (2002) Nature Biotech. 20: 1006 avoids poly(A) tailing and can be used in combination with tissue-specific promoters.

Methods for producing RNAi include chemical synthesis, in vitro transcription, digestion of long dsRNA by Dicer (in vitro or in vivo), in vivo expression from delivery vectors, and in vivo expression from PCR-derived RNAi expression cassettes (see, e.g., TechNotes 10(3) "Five Ways to Produce siRNAs," from Ambion, Inc., Austin TX; available at www.ambion.com).

Guidelines for designing siRNA molecules are available (see, for example, literature from Ambion, Inc., Austin TX; available at www.ambion.com). In a specific embodiment, the siRNA sequence has a G/C content of about 30-50%. In addition, if RNA polymerase III is used to transcribe the RNA, long stretches of more than four T or A residues are generally avoided. Online siRNA target finders are available, for example, from Ambion, Inc. (www.ambion.com), through the Whitehead Institute for Biomedical Research (www.jura.wi.mit.edu), or from Dharmacon Research, Inc. (www.dharmacon.com).

The antisense region of the RNAi molecule can be fully complementary to the target sequence, but does not need to be fully complementary as long as it specifically hybridizes to the target sequence (as defined above) and reduces the production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, hybridization of such an oligonucleotide to the target sequence can be carried out under reduced stringency, moderate stringency, or even stringent conditions as defined above.

In other embodiments, the antisense region of the RNAi has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or more sequence identity to the complement of the target sequence and reduces the production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the antisense region contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches compared to the target sequence. Mismatches are generally better tolerated at the ends of the dsRNA than in the central portion.

In a specific embodiment, RNAi is formed by intermolecular complexation between two separate sense and antisense molecules. RNAi comprises a ds region formed by intermolecular base pairs between the two separated strands. In other embodiments, RNAi comprises a ds region formed by intramolecular base pairing within a single nucleic acid molecule comprising the sense and antisense regions, typically an inverted repeat (e.g., shRNA or other stem-loop structures, or circular RNAi molecules). RNAi can also comprise a spacer between the sense and antisense regions.

RNAi molecules are usually highly selective. If desired, those skilled in the art can easily exclude candidate RNAi that may interfere with expression of nucleic acids other than the target by, for example, searching relevant databases to identify RNAi sequences that do not have substantial sequence homology with other known sequences using BLAST (available at www.ncbi.nlm.nih.gov/BLAST).

Kits for producing RNAi are commercially available from, for example, New England Biolabs, Inc. and Ambion, Inc.

Recombinant viral vectors can also contain heterologous nucleotide sequences that share homology with and recombine with sites on the host chromosome. This approach can be used to correct genetic defects in host cells.

The present invention also provides recombinant viral vectors expressing immunogenic polypeptides, for example, for use in vaccination. The heterologous nucleic acid can encode any immunogen of interest known in the art, including but not limited to immunogens from human immunodeficiency virus, influenza virus, gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like. Alternatively, the immunogen can be present in (e.g., incorporated into) or linked to (e.g., by covalent modification of) the viral capsid.

The use of parvovirus as a vaccine is known in the art (see, e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci. USA 91: 8507; U.S. Pat. No. 5,916,563 to Young et al., U.S. Pat. No. 5,905,040 to Mazzara et al., U.S. Pat. No. 5,882,652 to Samulski et al., U.S. Pat. No. 5,863,541 to Samulski et al.; the disclosures of which are incorporated herein by reference in their entireties). The antigen may be present in the viral capsid. Alternatively, the antigen may be expressed from a heterologous nucleic acid introduced into the genome of a recombinant vector.

The immunogenic polypeptide or immunogen can be any polypeptide suitable for protecting a subject from a disease, including but not limited to microbial, bacterial, protozoan, parasitic, fungal and viral diseases. For example, the immunogen can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein or an influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus (SIV) immunogen, or a human immunodeficiency virus (HIV) immunogen, such as HIV or SIV envelope GP160 protein, HIV or SIV matrix/capsid protein, and HIV or SIV gag, pol and env gene products). The immunogen can also be an arenavirus immunogen (e.g., a Lassa fever virus immunogen, such as a Lassa fever virus nucleocapsid protein gene and a Lassa fever envelope glycoprotein gene), a poxvirus immunogen (e.g., vaccinia, such as a vaccinia L1 or L8 gene), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen or a Marburg virus immunogen, such as the NP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFS viruses), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein gene, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen, or a severe acute respiratory syndrome (SARS) immunogen, such as the S [S1 or S2], M, E, or N protein, or an immunogenic fragment thereof). The immunogen may further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, or hepatitis C) immunogen, or any other vaccine immunogen known in the art.

Alternatively, the immunogen can be any tumor or cancer cell antigen. Alternatively, the tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are described in S.A. Rosenberg, (1999) Immunity 10:281). Illustrative cancer and tumor antigens include, but are not limited to, BRCA1 gene products, BRCA2 gene products, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124) including MART-1 (Coulie et al., (1991) J. Exp. Med. 180:35), gp100 (Wick et al., (1992) J. Exp. Med. 180:36), and pl5. al., (1988) J. Cutan. Pathol. 4:201) and MAGE antigens (MAGE-1, MAGE-2 and MAGE-3) (Van der Bruggen et al., (1991) Science, 254:1643), CEA, TRP-1; TRP-2; P-15 and tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neu gene product (U.S. Pat. No. 4,968,603); CA 125; HE4; LK26; FB5 (endosialin); TAG 72; AFP; CA19-9; NSE; DU-PAN-2; CA50; Span-1; CA72-4; HCG; STN (sialyl Tn antigen); c-erbB-2 protein; PSA; L-CanAg; estrogen receptor; milk fat globulin; p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62: 623); mucin antigen (International Patent Publication No. WO 90/05142); telomerase; nuclear matrix proteins; prostatic acid phosphatase; papillomavirus antigens; and antigens associated with melanoma, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, etc. (see, e.g., Rosenberg, (1996) Annu. Rev. Med. 47:481-91).

Alternatively, the heterologous nucleotide sequence can encode any polypeptide that is desired to be produced in cells in vitro, ex vivo or in vivo. For example, a viral vector can be introduced into cultured cells and the expressed protein product isolated therefrom.

Those skilled in the art will appreciate that the heterologous polynucleotide of interest can be operably associated with appropriate control sequences. For example, the heterologous nucleic acid can be operably associated with expression control elements (such as transcription/translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, enhancers, etc.).

Those skilled in the art will further appreciate that various promoter/enhancer elements can be used, depending on the desired level and tissue-specific expression. Depending on the desired expression pattern, the promoter/enhancer can be constitutive or inducible. The promoter/enhancer can be native or foreign, and can be a natural or synthetic sequence. "Foreign" means that the transcription initiation region is not found in the wild-type host into which it is introduced.

The promoter/enhancer element can be native to the target cell or subject to be treated and/or native to the heterologous nucleic acid sequence. The promoter/enhancer element is generally selected so that it is functional in the target cell of interest. In representative embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element. The promoter/enhancer element can be constitutive or inducible.

Inducible expression control elements are generally used in those applications where the regulation of heterologous nucleic acid sequence expression needs to be provided. The inducible promoter/enhancer element for gene delivery can be tissue-specific or tissue-preferred promoter/enhancer element, and include muscle specificity or preference (including heart, skeleton and/or smooth muscle), neural tissue specificity or preference (including brain specificity), eyes (including retina specificity and cornea specificity), liver specificity or preference, bone marrow specificity or preference, pancreas specificity or preference, spleen specificity or preference and lung specificity or preference promoter/enhancer element. Other inducible promoter/enhancer elements include hormone inducible and metal inducible elements. Exemplary inducible promoter/enhancer elements include but are not limited to Tet on/off element, RU486 inducible promoter, ecdysone inducible promoter, rapamycin inducible promoter and metallothionein promoter.

In embodiments where a heterologous nucleic acid sequence is transcribed and translated in the target cell, specific initiation signals are generally used for efficient translation of the inserted protein coding sequence. These exogenous translation control sequences, which may include the ATG start codon and adjacent sequences, may be of a variety of origins (natural and synthetic).

The present invention also provides a method for producing the viral vector of the present invention. In representative embodiments, the present invention provides a method for producing a recombinant viral vector, the method comprising providing (a) a template to a cell in vitro, the template comprising (i) a polynucleotide of interest and (ii) a packaging signal sequence (e.g., one or more (e.g., two) terminal repeats, e.g., AAV terminal repeats) sufficient to encapsulate the AAV template into viral particles, and (b) an AAV sequence (e.g., AAV rep and AAV cap sequence) sufficient to replicate the template and encapsulate the template into viral particles. Under conditions such that recombinant viral particles comprising a template packaged in a capsid are produced in the cell, the template and AAV replication and capsid sequences are provided. The method may also include a step of collecting viral particles from the cell. Viral particles can be collected from culture medium and/or by lysing cells.

In an illustrative embodiment, the present invention provides a method for producing rAAV particles comprising an AAV capsid, the method comprising: providing to cells in vitro a nucleic acid encoding an AAV capsid, an AAV rep coding sequence, an AAV vector genome comprising a polynucleotide of interest, and helper functions for generating a productive AAV infection; and allowing assembly of AAV particles comprising the AAV capsid and encapsidating the AAV vector genome.

The cells are typically cells that are permissive for AAV viral replication. Any suitable cell known in the art, such as mammalian cells, may be used. Also suitable are trans-complementing packaging cell lines that provide functions deleted from the replication-defective helper virus, such as 293 cells or other E1a trans-complementing cells.

AAV replication and capsid sequences can be provided by any method known in the art. Current protocols typically express the AAV rep/cap genes on a single plasmid. It is not necessary to provide the AAV replication and packaging sequences together, although it may be convenient to do so. The AAV rep and/or cap sequences can be provided by any viral or non-viral vector. For example, the rep/cap sequences can be provided by a hybrid adenovirus or herpes virus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus vector). EBV vectors can also be used to express the AAV cap and rep genes. One advantage of this approach is that EBV vectors are episomal, but will maintain a high copy number throughout successive cell divisions (i.e., stably integrated into the cell as an extrachromosomal element, designated as an EBV-based nuclear episome).

As another alternative, the rep/cap sequences can be stably harbored within the cell (either episomal or integrated).

Typically, the AAV rep/cap sequences are not flanked by AAV packaging sequences (eg, AAV ITRs) to prevent rescue and/or packaging of these sequences.

The template (e.g., rAAV vector genome) can be provided to the cell using any method known in the art. For example, the template can be provided by a non-viral (e.g., plasmid) or viral vector. In a specific embodiment, the template is provided by a herpes virus or adenovirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus). As another illustration, Palombo et al., (1998) J. Virol. 72: 5025 describes a baculovirus vector carrying a reporter gene flanked by AAV ITRs. EBV vectors can also be used to deliver the template, as described above for the rep/cap gene.

In another representative embodiment, the template is provided by a replicating rAAV virus. In yet other embodiments, the AAV provirus is stably integrated into the chromosome of the cell.

In order to obtain maximum viral titers, cells are typically provided with helper virus functions (e.g., adenovirus or herpes virus) that are important for productive AAV infection. The helper virus sequences required for AAV replication are known in the art. Typically, these sequences are provided by a helper adenovirus or herpes virus vector. Alternatively, the adenovirus or herpes virus sequences can be provided by another non-viral or viral vector, for example, as a non-infectious adenovirus miniplasmid carrying all the helper genes required for efficient AAV production, as described in Ferrari et al., (1997) Nature Med. 3: 1295 and U.S. Patent Nos. 6,040,183 and 6,093,570.

In addition, helper virus function can be provided by packaging cells that have helper genes integrated into the chromosome or maintained as stable extrachromosomal elements. In representative embodiments, the helper virus sequences are not packaged in AAV virions, e.g., are not flanked by AAV ITRs.

Those skilled in the art will appreciate that it may be advantageous to provide the AAV replication and capsid sequences and the helper virus sequences (e.g., adenovirus sequences) on a single helper construct. The helper construct may be a non-viral or viral construct, but may alternatively be a hybrid adenovirus or hybrid herpes virus containing the AAV rep/cap genes.

In one embodiment, the AAV rep/cap sequence and the adenoviral helper sequence are provided by a single adenoviral helper vector. The vector also contains a rAAV template. The AAV rep/cap sequence and/or the rAAV template can be inserted into a deleted region of the adenovirus (e.g., the E1a or E3 region).

In another embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. The rAAV template is provided as a plasmid template.

In another illustrative embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector maintained in the cell as an extrachromosomal element (e.g., as an "EBV-based nuclear episome," see Margolski, (1992) Curr. Top. Microbiol. Immun. 158: 67).

In another exemplary embodiment, the AAV rep/cap sequence and adenovirus helper sequence are provided by a single adenovirus helper. The rAAV template is provided as a separate replicating viral vector. For example, the rAAV template can be provided by rAAV particles or a second recombinant adenovirus particle.

According to the above method, the hybrid adenoviral vector generally comprises adenoviral 5' and 3' cis sequences (i.e., adenoviral terminal repeats and PAC sequences) sufficient for adenoviral replication and packaging. The AAV rep/cap sequence and (if present) the rAAV template are embedded in the adenoviral backbone and are flanked by the 5' and 3' cis sequences so that these sequences can be packaged into the adenoviral capsid. As described above, in representative embodiments, the adenoviral helper sequence and the AAV rep/cap sequence are not flanked by AAV packaging sequences (e.g., AAV ITRs) so that these sequences are not packaged into AAV virions.

Herpes viruses can also be used as helper viruses in AAV packaging methods. Hybrid herpes viruses encoding the AAV rep protein can advantageously facilitate more scalable AAV vector production protocols. Hybrid herpes simplex virus type 1 (HSV-1) vectors expressing the AAV-2 rep and cap genes have been described (Conway et al., (1999) Gene Therapy 6:986 and WO 00/17377, the disclosures of which are incorporated herein in their entirety).

As another alternative, the viral vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the rep/cap genes and the rAAV template as described by Urabe et al., (2002) Human Gene Therapy 13: 1935-43.

Other methods of producing AAV use stably transformed packaging cells (see, eg, US Pat. No. 5,658,785).

AAV vector stocks without contaminating helper viruses can be obtained by any method known in the art. For example, AAV and helper viruses can be easily distinguished based on size. AAV can also be separated from helper viruses based on affinity for a heparin substrate (Zolotukhin et al., (1999) Gene Therapy 6:973). In a representative embodiment, a deleted replication-deficient helper virus is used so that any contaminating helper virus does not have the ability to replicate. As another alternative, an adenoviral helper lacking late gene expression can be used, since only adenoviral early gene expression is required to mediate the packaging of the AAV virus. Adenoviral mutants deficient in late gene expression are known in the art (e.g., ts100K and ts149 adenoviral mutants).

The packaging methods of the present invention can be used to generate high titer viral particle stocks. In specific embodiments, the viral stock has a titer of at least about 10 ⁵ transducing units (tu)/ml, at least about 10 ⁶ tu/ml, at least about 10 ⁷ tu/ml, at least about 10 ⁸ tu/ml, at least about 10 ⁹ tu/ml, or at least about 10 ¹⁰ tu/ml.

In a specific embodiment, the present invention provides a pharmaceutical composition comprising a viral vector of the present invention and optionally other medical agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc., in a pharmaceutically acceptable carrier. For injection, the carrier is typically a liquid. For other modes of administration, the carrier can be a solid or a liquid. For administration by inhalation, the carrier will be respirable and will preferably be in the form of solid or liquid particles.

The term "pharmaceutically acceptable" refers to a substance that is not toxic or otherwise undesirable, ie, a substance that can be administered to a subject without causing any undesirable biological effects.

One aspect of the present invention is a method for transferring a polynucleotide of interest to a cell in vitro. Viral vectors can be introduced into cells at an appropriate multiplicity of infection according to standard transduction methods suitable for specific target cells. The titer of the viral vector or capsid used for administration can vary depending on the type and number of target cells and the specific viral vector or capsid, and can be determined by those skilled in the art without excessive experimentation. In a specific embodiment, at least about 10 ³ infectious units, more preferably at least about 10 ⁵ infectious units, are introduced into the cells.

The cell that can introduce viral vector can be any type, including but not limited to neural cell (comprising cells of peripheral and central nervous system, particularly, brain cell such as neuron, oligodendrocyte, neuroglia, astrocyte), lung cell, eye cell (comprising retinal cell, retinal pigment epithelium and keratocyte), epithelial cell (such as intestinal and respiratory epithelial cell), skeletal muscle cell (comprising myoblast, myotube and myofiber), diaphragm muscle cell, dendritic cell, pancreatic cell (comprising islet cell), hepatocyte, gastrointestinal cell (comprising smooth muscle cell, epithelial cell), heart cell (comprising cardiomyocyte), osteocyte (such as bone marrow stem cell), hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, joint cell (comprising such as cartilage, meniscus, synovium and bone marrow), germ cell etc.Alternately, cell can be any progenitor cell.As additional selection, cell can be stem cell (such as neural stem cell, hepatic stem cell).As another alternative, cell can be cancer or tumor cell (cancer and tumor as described above). Furthermore, as stated above, the cells may be from any species of origin.

Viral vectors can be introduced into cells in vitro, for the purpose of administering modified cells to a subject. In a specific embodiment, cells are removed from a subject, viral vectors are imported thereto, and then the cells are replaced back into the subject. Cells are taken out from a subject for in vitro treatment and then introduced back into the subject's method, which is known in the art (see, for example, U.S. Patent number 5,399,346). Alternatively, recombinant viral vectors are introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need.

Suitable cells for ex vivo gene therapy are described above. The dosage of cells administered to a subject will vary depending on the age, condition, and species of the subject, the cell type, the nucleic acid expressed by the cells, the mode of administration, and the like. Typically, at least about 10 ² to about 10 ⁸ or about 10 ³ to about 10 ⁶ cells per dose are administered in a pharmaceutically acceptable carrier. In specific embodiments, cells transduced with a viral vector are administered to a subject in an effective amount in combination with a pharmaceutical carrier.

In some embodiments, cells that have been transduced with viral vectors can be administered to elicit an immunogenic response to the delivered polypeptide (e.g., expressed as a transgene or in a capsid). Typically, a certain amount of cells expressing an effective amount of polypeptide is administered in combination with a pharmaceutically acceptable carrier. Alternatively, the dosage is sufficient to produce a protective immune response (as defined above). As long as the benefits of administering the immunogenic polypeptide outweigh any of its shortcomings, the degree of protection conferred does not need to be complete or permanent.

Another aspect of the present invention is a method of administering a viral vector of the present invention to a subject. In a specific embodiment, the method comprises a method of delivering a polynucleotide of interest to an animal subject, the method comprising administering to the animal subject an effective amount of a viral vector according to the present invention. The viral vector of the present invention can be administered to a human subject or animal in need thereof by any method known in the art. Alternatively, the viral vector is delivered in an effective dose in a pharmaceutically acceptable carrier.

The viral vectors of the present invention can be further administered to a subject to elicit an immunogenic response (e.g., as a vaccine). Typically, the vaccine of the present invention comprises an effective amount of virus in combination with a pharmaceutically acceptable carrier. Alternatively, the dosage is sufficient to produce a protective immune response (as defined above). As long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages, the degree of protection conferred does not need to be complete or permanent. The subject and immunogen are as described above.

The dosage of the viral vector to be administered to a subject will depend on the mode of administration, the disease or condition to be treated, the condition of the individual subject, the specific viral vector and the nucleic acid to be delivered, and can be determined in a routine manner. Exemplary dosages for achieving a therapeutic effect are at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ³ , 10 ¹⁴ , 10 ¹⁵ transducing units or higher viral titers, preferably about 10 ⁷ or 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ or 10 ¹⁴ transducing units, and even more preferably about 10 ¹² transducing units.

In specific embodiments, more than one administration (e.g., two, three, four, or more administrations) may be used to achieve desired levels of gene expression over various intervals (e.g., daily, weekly, monthly, yearly, etc.).

Exemplary routes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., by aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraocular), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to bones, diaphragm, and/or myocardium], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., skin and mucosal surfaces, including airway surfaces and transdermal administration), intralymphatic, and the like, as well as direct tissue or organ injection (e.g., liver, skeletal muscle, myocardium, diaphragm muscle, or brain). Administration to tumors (e.g., in or near a tumor or lymph node) is also possible. The most appropriate route in any given case will depend on the nature and severity of the condition being treated and the nature of the particular vector being used.

Delivery to any one of these tissues can also be achieved by delivering a depot comprising a viral vector, which can be implanted in the tissue or which can be contacted with a membrane or other matrix comprising the viral vector. The example of such an implantable matrix or substrate is described in U.S. Patent number 7,201,898.

The present invention can be used for treating the disease of tissue or organ.Alternatively, the present invention can be implemented to deliver nucleic acid to tissue or organ, described tissue or organ is used as for producing and circulates in blood or systemically is delivered to other tissues to treat disease (for example metabolic disorder, such as diabetes (for example insulin), hemophilia (for example factor IX or factor VIII) or lysosomal storage disorder (such as Gaucher's disease [glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase] or Fabry disease [α-galactosidase A]) or glycogen storage disease (such as Pompe disease [lysosomal acid α-glucosidase]) protein product (for example enzyme) or non-translated RNA (for example RNAi, microRNA, antisense RNA) platform.Other suitable proteins for treating metabolic disorder are as described above.

Injectables can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or as emulsions. Alternatively, viral vectors can be administered in a local rather than systemic manner, for example in the form of a reservoir or sustained-release formulation. In addition, viral vectors can be delivered dry to surgical implantable matrices, such as bone graft substitutes, sutures, stents, etc. (e.g., as described in U.S. Patent No. 7,201,898).

Pharmaceutical compositions suitable for oral administration can be present in discrete units, such as capsules, cachets, lozenges or tablets, each containing a predetermined amount of the composition of the present invention; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as a water-in-oil or water-in-oil emulsion. Oral delivery can be performed by compounding the viral vector of the present invention with a carrier that can withstand degradation by digestive enzymes in the animal intestine. Examples of such carriers include plastic capsules or tablets known in the art. These preparations are prepared by any suitable pharmaceutical method, including the step of combining the composition with a suitable carrier (the carrier may contain one or more auxiliary ingredients as described above). Generally, pharmaceutical compositions according to embodiments of the present invention are prepared by uniformly and intimately mixing the composition with a liquid or finely divided solid carrier or both, and then shaping the resulting mixture if necessary. For example, tablets can be prepared by compressing or molding a powder or granules containing the composition and, optionally, one or more auxiliary ingredients. Compressed tablets are prepared by compressing a free-flowing composition (such as a powder or granules optionally mixed with a binder, lubricant, inert diluent and/or surfactant/dispersant) in a suitable machine. Molded tablets are made by molding in a suitable machine the powdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sublingual) administration include lozenges comprising the compositions of this invention in a flavored base, usually sucrose and acacia or tragacanth, and pastilles comprising the compositions in an inert base such as gelatin and glycerin or sucrose and acacia.

Pharmaceutical compositions suitable for parenteral administration may comprise sterile aqueous and non-aqueous injections of the compositions of the present invention, which are optionally isotonic with the blood of the intended recipient. These preparations may contain antioxidants, buffers, antibacterials, and solutes that make the compositions isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions, and emulsions may include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol/water solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oil. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present.

The compositions may be presented in unit/dose or multi-dose containers, for example in sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, for example saline or water for injection, immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the above-mentioned types. For example, the injectable, stable, sterile compositions of the present invention can be provided in unit dosage forms in sealed containers. The compositions can be provided in the form of lyophilizates that can be reconstituted with suitable pharmaceutically acceptable carriers to form liquid compositions suitable for injection into a subject. The unit dosage form can be from about 1 μg to about 10g of the compositions of the present invention. When the composition is substantially insoluble in water, a sufficient amount of a physiologically acceptable emulsifier can be included in an aqueous carrier in sufficient amounts to emulsify the composition. One such useful emulsifier is phosphatidylcholine.

Pharmaceutical compositions suitable for rectal administration may be presented as unit-dose suppositories. These may be prepared by mixing the composition with one or more conventional solid carriers (eg, cocoa butter) and shaping the resulting mixture.

The pharmaceutical composition of the present invention suitable for topical application to the skin can be in the form of an ointment, cream, lotion, paste, gel, spray, aerosol or oil. Useful carriers include, but are not limited to, vaseline, lanolin, polyethylene glycol, alcohol, transdermal enhancers and combinations of two or more thereof. In some embodiments, for example, topical delivery can be performed by mixing the pharmaceutical composition of the present invention with a lipophilic agent (e.g., DMSO) that can penetrate the skin.

Pharmaceutical compositions suitable for transdermal administration can be in the form of a dispersible patch, which is suitable for maintaining close contact with the subject's epidermis for a long time. Compositions suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharm. Res. 3: 318 (1986)) and are generally in the form of an aqueous solution of the composition of the present invention with optional buffering. Suitable formulations can include citrate or bis\tris buffer (pH 6) or ethanol/water and can contain 0.1 to 0.2 M of the active ingredient.

The viral vectors disclosed herein can be administered to the lungs of a subject by any suitable means, for example, by administering an aerosol suspension of respirable particles composed of the viral vector, which is inhaled by the subject. The inhalable particles can be liquid or solid. As known to those skilled in the art, an aerosol of liquid particles containing the viral vector can be produced by any suitable device, such as a pressure-driven nebulizer or ultrasonic nebulizer. See, for example, U.S. Patent No. 4,501,729. An aerosol of solid particles containing the viral vector can also be produced by any solid particulate pharmaceutical aerosol generator using techniques known in the pharmaceutical field.

Production of the Factor VIII protein of the present invention

Many expression vectors can be used to create genetically engineered cells. Some expression vectors are designed to express large amounts of recombinant protein after expansion of transfected cells under various conditions that favor the selection of high-expressing cells. Some expression vectors are designed to express large amounts of recombinant protein without the need for expansion under selective pressure. The present invention encompasses the production of genetically engineered cells according to standard methods in the art and does not rely on the use of any specific expression vector or expression system.

In order to generate genetically engineered cells to produce large amounts of FVIII protein, cells are transfected with expression vectors containing polynucleotides (e.g., cDNA) encoding the protein. In some embodiments, the FVIII protein is expressed with a co-transfected enzyme selected that results in appropriate post-translational modifications of the FVIII protein in a given cell system.

The cells can be derived from a variety of sources, but in other aspects can be cells transfected with an expression vector containing a nucleic acid molecule (eg, cDNA) encoding a FVIII protein.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art and are fully described in the literature. See, e.g., Sambrook, et al., Molecular Cloning; A Laboratory Manual, 2nd ed. (1989); DNA Cloning , Volumes I and II (D. N. Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds., 1984); Transcription and Translation (B. D. Hames & S. J. Higgins, eds., 1984); Animal Cell Culture (R. I. Freshney, ed., 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Academic Press, Inc.), particularly Volumes 154 and 155 (edited by Wu and Grossman and Wu, respectively); Gene Transfer Vectors for Mammalian Cells. (JH Miller and MP Calos, eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology , Mayer and Walker, eds. (Academic Press, London, 1987); Scopes, Protein Purification: Principles and Practice , 2nd ed., 1987 (Springer-Verlag, NY); and Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell, eds., 1986). The entire contents of all patents, patent applications, and publications cited in this specification are incorporated herein by reference.

genetic engineering technology

The production of cloned genes, recombinant DNA, vectors, transformed cells, proteins and protein fragments by genetic engineering is well known. See, for example, U.S. Patent No. 4,761,371 to Bell et al. at column 6, line 3 to column 9, line 65; U.S. Patent No. 4,877,729 to Clark et al. at column 4, lines 38 to 76; U.S. Patent No. 4,912,038 to Schilling at column 3, line 26 to column 14, line 12; and U.S. Patent No. 4,879,224 to Wallner at column 6, line 8 to column 8, line 59.

Vector (vector) is a reproducible DNA construct. Vector is used to increase the nucleic acid of coding FVIII protein and/or express the nucleic acid of coding FVIII protein in this article. Expression vector is a reproducible nucleic acid construct, wherein the nucleotide sequence of coding FVIII protein is operably connected to a suitable control sequence, and this control sequence can realize the expression of nucleotide sequence to produce FVIII protein in a suitable host cell. The needs of this control sequence will change according to the selected host cell and the selected transformation method. Usually, control sequence includes transcription promoter, the optional operator sequence for controlling transcription, the sequence of encoding suitable mRNA ribosome bind site and the sequence of control transcription and translation termination.

Vectors include plasmids, viruses (e.g., AAV, adenovirus, cytomegalovirus), phages, and integrable DNA segments (i.e., segments that can be integrated into the host cell genome by recombination). Vectors can replicate and function independently of the host cell genome (e.g., by transient expression), or can be integrated into the host cell genome itself (e.g., stably integrated). Expression vectors can contain a promoter and an RNA binding site that are operably linked to the nucleic acid molecule to be expressed and can operate in the host cell and/or organism.

When DNA regions or nucleotide sequences are functionally related to each other, they are operably linked or operably associated. For example, if a promoter controls the transcription of a coding sequence, then a promoter is operably linked to the coding sequence; or if a ribosome binding site is positioned to allow translation of the coding sequence, then the ribosome binding site is operably linked to the coding sequence.

Suitable host cells include prokaryotes, yeast or higher order eukaryotic cells such as mammalian cells and insect cells. Cells derived from multicellular organisms are particularly suitable hosts for recombinant FVIII protein synthesis, and mammalian cells are particularly preferred. The propagation of such cells in cell culture has become routine ( Tissue Culture , Academic Press, Kruse and Patterson, eds. (1973)). Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines and WI138, HEK 293, BHK, COS-7, CV and MDCK cell lines. The expression vectors of such cells typically include (if necessary) an origin of replication, a promoter upstream of the nucleotide sequence encoding the FVIII protein to be expressed and operably associated therewith, and a ribosome binding site, an RNA splice site (if using intron-containing genomic DNA), a polyadenylation site and a transcription termination sequence. In one embodiment, expression can be performed in Chinese Hamster Ovary (CHO) cells using the expression system of US Patent No. 5,888,809, which is incorporated herein by reference in its entirety.

Transcriptional and translational control sequences in expression vectors used in transformed vertebrate cells are typically provided by viral sources. Non-limiting examples include promoters derived from polyoma virus, adenovirus 2, and simian virus 40 (SV40). See, for example, U.S. Patent No. 4,599,308.

The vector may be constructed to include an exogenous origin of replication, such as one derived from SV 40 or other viral (e.g., polyoma virus, adenovirus, VSV, or BPV) sources, or the host cell chromosomal replication machinery may provide an origin of replication. If the vector is integrated into the host cell chromosome, the host cell chromosome is usually sufficient.

In addition to using vectors containing viral origins of replication, mammalian cells can also be transformed by co-transformation with a nucleic acid molecule encoding the FVIII protein using a selective marker. Non-limiting examples of suitable selective markers are dihydrofolate reductase (DHFR) or thymidine kinase. This method is further described in U.S. Patent No. 4,399,216, which is incorporated herein by reference in its entirety.

Other methods suitable for adaptation to synthesize FVIII protein in recombinant vertebrate cell culture include those described in Gething et al. Nature 293:620 (1981); Mantei et al. Nature 281:40; and Levinson et al., EPO Application Nos. 117,060A and 117,058A, each of which is incorporated herein by reference in its entirety.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as baculovirus expression vectors (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) can be used to practice the present invention, as described in U.S. Patent Nos. 4,745,051 and 4,879,236 to Smith et al. Generally, baculovirus expression vectors comprise a baculovirus genome containing the nucleotide sequence to be expressed, inserted into the polyhedrin gene at a position within the range of the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of the baculovirus polyhedrin promoter.

Prokaryotic host cells include gram-negative or gram-positive organisms, such as Escherichia coli (E. coli) or rod-shaped bacteria. Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Exemplary bacterial host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), and E. coli 294 (ATCC 31,446). A variety of suitable prokaryotic and microbial vectors can be used. E. coli is typically transformed using pBR322. The most commonly used promoters in recombinant microbial expression vectors include the β-lactamase (penicillinase) and lactose promoter systems (Chang et al. Nature 275: 615 (1978); and Goeddel et al. Nature 281: 544 (1979)), the tryptophan (trp) promoter system (Goeddel et al. Nucleic Acids Res. 8: 4057 (1980) and EPO Application Publication No. 36,776), and the tac promoter (De Boer et al. Proc. Natl. Acad. Sci. USA 80: 21 (1983)). The promoter and Shine-Dalgarno sequence (for prokaryotic expression) are operably linked to the nucleic acid encoding the FVIII protein, i.e., they are positioned to promote transcription of the FVIII messenger RNA from the DNA.

Eukaryotic microorganisms such as yeast cultures can also be transformed with protein encoding vectors (see, e.g., U.S. Patent No. 4,745,057). Saccharomyces cerevisiae is the most commonly used of the lower eukaryotic host microorganisms, although many other strains are also commonly available. Yeast vectors can contain an origin of replication or an autonomously replicating sequence (ARS) from a 2-micron yeast plasmid, a promoter, a nucleic acid encoding the FVIII protein, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7 (Stinchcomb et al. Nature 282:39 (1979); Kingsman et al. Gene 7:141 (1979); Tschemper et al. Gene 10:157 (1980)). Suitable promoter sequences for yeast vectors include the promoter for metallothioneins, the promoter for 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem. 255:2073 (1980), or the promoters for other glycolytic enzymes (Hess et al. J. Adv. Enzyme Reg. 7:149 (1968); and Holland et al. Biochemistry 17:4900 (1978)). Vectors and promoters suitable for yeast expression are further described in R. Hitzeman et al., EPO Publication No. 73,657.

The cloned coding sequence of the present invention can encode FVIII of any species, including mouse, rat, dog, opossum, rabbit, cat, pig, horse, sheep, cow, guinea pig, opossum, platypus and human, but preferably encodes human FVIII protein. Nucleic acids encoding FVIII that can hybridize with the nucleic acids encoding the protein disclosed herein are also included. Such sequences can be hybridized with nucleic acids encoding FVIII proteins disclosed herein under reduced stringency conditions or even under stringent conditions (e.g., stringent conditions represented by washing stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60°C or even 70°C) in standard in situ hybridization assays. See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Edition 1989) Cold Spring Harbor Laboratory).

The FVIII protein produced according to the present invention can be expressed in transgenic animals by known methods. See, for example, U.S. Patent No. 6,344,596, the entire contents of which are incorporated herein by reference. In short, transgenic animals can include, but are not limited to, farm animals (e.g., pigs, goats, sheep, cows, horses, rabbits, etc.), rodents (e.g., mice, rats, and guinea pigs), and domestic pets (e.g., cats and dogs). In some embodiments, livestock such as pigs, sheep, goats, and cows are particularly preferred.

The transgenic animals of the present invention are produced by introducing a suitable polynucleotide encoding a human FVIII protein of the present invention into a one-cell embryo in such a manner that the polynucleotide is stably integrated into the DNA of the germline cells of the mature animal and inherited in a normal Mendelian manner. The transgenic animals of the present invention will have a phenotype of producing FVIII protein in body fluids and/or tissues. The FVIII protein can be removed from these fluids and/or tissues and processed, for example, for therapeutic use. (See, for example, Clark et al. "Expression of human anti-hemophilic factor IX in the milk of transgenic sheep" Bio/Technology 7: 487-492 (1989); Van Cott et al. "Haemophilic factors produced by transgenic livestock: abundance can enable alternative therapies worldwide" Haemophilia 10(4): 70-77 (2004), the entire contents of which are incorporated herein by reference).

DNA molecules can be introduced into embryos in a variety of ways, including but not limited to microinjection, calcium phosphate-mediated precipitation, liposome fusion, or retroviral infection of totipotent or pluripotent stem cells. The transformed cells are then introduced into the embryo and incorporated therein to form a transgenic animal. For example, methods for making transgenic animals are described in L.M. Houdebine's Transgenic Animal Generation and Use , Harwood Academic Press, 1997. Transgenic animals can also be produced using methods of nuclear transfer or cloning of embryonic or adult cell lines, as described, for example, in Campbell et al., Nature 380: 64-66 (1996) and Wilmut et al., Nature 385: 810-813 (1997). In addition, as described in U.S. Patent No. 5,523,222, techniques utilizing cytoplasmic injection of DNA can be used.

Transgenic animals producing FVIII can be obtained by introducing a chimeric construct comprising a FVIII coding sequence. Methods for obtaining transgenic animals are well known. See, e.g., Hogan et al., MANIPULATING THE MOUSE EMBRYO , (Cold Spring Harbor Press 1986); Krimpenfort et al., Bio/Technology 9:88 (1991); Palmiter et al., Cell 41:343 (1985), Kraemer et al., GENETIC MANIPULATION OF THE EARLY MAMMALIAN EMBRYO, (Cold Spring Harbor Laboratory Press 1985); Hammer et al., Nature 315:680 (1985); Wagner et al., U.S. Pat. No. 5,175,385; Krimpenfort et al., U.S. Pat. No. 5,175,384, Janne et al., Ann. Med. 24:273 (1992), Bremet et al., Cell 41:343 (1985), Kraemer et al., GENETIC MANIPULATION OF THE EARLY MAMMALIAN EMBRYO, (Cold Spring Harbor Laboratory Press 1985); Hammer et al., Nature 315:680 (1985); Wagner et al., U.S. Pat. No. 5,175,385; Krimpenfort et al., U.S. Pat. No. 5,175,384, Janne et al., Ann. Med. 24:273 (1992), Bremet et al., Cell 41:343 (1985), Kraemer et al., GENETIC MANIPULATION OF THE EARLY MAMMALIAN EMBRYO al., Chim. Oggi. 11:21 (1993), Clark et al., U.S. Patent No. 5,476,995, the entire contents of which are incorporated herein by reference.

In some embodiments, cis-acting regulatory regions that are "active" in mammary tissue may be used because, under the physiological conditions of synthetic milk, the promoter is more active in mammary tissue than in other tissues. These promoters include, but are not limited to, the short and long whey acid protein (WAP), the short and long α, β, and κ caseins, α-lactalbumin, and β-lactoglobulin ("BLG") promoters. Signal sequences that direct secretion of the expressed protein into other body fluids, particularly blood and urine, may also be used in accordance with the present invention. Examples of these sequences include signal peptides for secreted coagulation factors, including FVIII, protein C, and tissue plasminogen activator.

In addition to the promoters discussed above, useful sequences that regulate transcription are enhancers, splicing signals, transcription termination signals, polyadenylation sites, buffering sequences, RNA processing sequences, and other sequences that regulate transgene expression.

Preferably, the expression system or construct comprises a 3' non-translated region downstream of the nucleotide sequence encoding the desired recombinant protein. This region can increase transgenic expression. In this respect, a useful 3' non-translated region is a sequence that provides a poly A signal.

Suitable heterologous 3'-untranslated sequences can be derived from, for example, the SV40 small t antigen, the casein 3' untranslated region, or other 3' untranslated sequences well known in the art. Ribosome binding sites are also important for increasing the expression efficiency of FVIII. Similarly, sequences that regulate post-translational modification of FVIII are useful in the present invention.

Having described the present invention, it will be explained in more detail in the following examples, which are included herein for illustrative purposes only and are not intended to limit the present invention.

Example 1

synthetic liver-specific promoter

We designed and fully synthesized a number of artificial promoters containing conserved basal promoter elements and transcription start sites. The basal promoters were linked to a number of liver-specific transcription factor binding sites at their 5' termini for liver-specific expression. The promoter, designated LXP3.3 (Figure 1) (SEQ ID NO: 1), was chosen due to its small size (200 bp) and high activity in the human hepatoma cell line Huh7 in vitro using a luciferase reporter gene and transfection assays.

The LXP3.3 promoter was then further tested using a LacZ reporter gene packaged in an AAV9 vector, which has broad tissue tropism in the liver, heart, and muscle, among others. Parallel in vivo experiments were performed to compare promoter activity and liver specificity with the strong and liver-specific promoter thyroxine-binding globulin (TBG), previously reported as one of the strongest liver-specific promoters in both small and large animal models. AAV9 vectors containing either the LXP3.3-LacZ or TBG-LacZ expression cassettes were injected into C56/B6 mice via the tail vein at two different doses. As shown in Figure 2A, X-gal staining of the liver and heart revealed robust liver expression and lack of cardiac expression for both promoters. Quantitative analysis of tissue homogenates revealed that, despite the LXP3.3 promoter being only 200 bp in size and the TBG promoter being 681 bp in size, both promoters achieved nearly identical LacZ expression levels and tissue specificity for the liver (Figure 2B). Specifically, quantitative LacZ enzyme activity analysis revealed that for both the LXP3.3 and TBG promoters, gene expression in the liver was more than 300-fold higher than in the heart. Furthermore, in the liver, LacZ enzyme activity achieved with the liver-specific promoter was 500-fold higher than that achieved with the ubiquitous CMV promoter (Figure 2C). On the other hand, in the heart, the CMV promoter achieved LacZ expression nearly 13-fold higher than that achieved with the liver-specific promoter (Figure 2B). These results demonstrate that the synthetic promoter LXP3.3 is highly active and specific in the liver.

Example 2

Synthetic promoter intron cassette

The fully synthetic promoter LXP3.3 is linked at its 3' end to a small intron of VH4 with a short non-natural exon junction sequence. This intron was initially tested by in vitro transfection experiments using a weak promoter driving the BDD human FVIII gene. The addition of the VH4 intron provides higher gene expression than promoters without introns, and also provides higher expression than the commonly used chimeric intron CIN (Promega) (Figure 3). Therefore, we combined the LXP3.3 promoter and the VH4 intron with an artificial exon junction sequence and named it LXP3.3I (SEQ ID NO: 2). To test its activity in driving FVIII expression, we inserted the fully synthetic promoter LXP3.3I upstream of the human BDD-deleted FVIII gene (SEQ ID NO: 3), followed by a small polyadenylation site. Subsequently, the entire gene expression cassette (SEQ ID NO: 4) was cloned into an AAV vector plasmid backbone with two AAV inverted terminal repeats for vector DNA replication and vector genome packaging ( FIG. 4 ).

To compare promoter activity in vitro, the ubiquitous CMV promoter linked to the SV40 intron (a commonly used powerful combination) was used to replace LXP3.3I in the FVIII expression cassette. After the plasmid was transfected into the human hepatoma cell line Huh7, FVIII activity was determined using a chromogenic kit. As shown in the transfection experiment in Figure 5, LXP3.3I produced higher FVIII activity in cell culture medium than the CMV promoter. In addition, we also compared a fully synthetic human BDD-deleted FVIII gene with a BDD FVIII gene of the natural human DNA sequence in transfection experiments. When driven by the same LXP3.3I, the two genes encoding the same FVIII amino acid sequence but using different codon selection did not show significant differences in FVIII activity in human cells (Figure 6). However, when a different liver-specific promoter was used, the BDD FVIII gene with native codons showed lower expression compared to the BDD FVIII gene driven by LXP3.3I, indicating that the higher gene expression was mainly due to the LXP3.3I promoter.

We next tested in vivo gene expression activity in a commonly used FVIII gene knockout hemophilia A mouse model. The LXP3.3I-hF8 gene expression cassette was packaged into an AAV9 capsid and injected into the tail vein of FVIII KO mice. Two different doses of vectors (2x 10 ¹¹ and 4x 10 ¹⁰ vector genomes per mouse) were used to examine in vivo expression. As shown in Figure 7A, at these two doses, high levels and long-term gene expression were achieved over a period of more than 1 year after vector administration. In addition to the colorimetric assay for FVIII activity, ELISA was also used to examine the amount of FVIII protein secreted in plasma (Figure 7B). The results showed that, compared to the reference standard of full-length wild-type human FVIII protein, BDD FVIII protein concentration was approximately 50% lower than the reading obtained by the colorimetric assay for FVIII activity relative to the same full-length human FVIII. This difference (ELISA reading lower than FVIII activity reading) may be due to lacking long B domain in BDD FVIII and causing, and it almost comprises half of wild-type FVIII length.Because ELISA kit uses the polyclonal antibody for the wild-type full-length FVIII that comprises B domain, it is expected that the BDD FVIII lacking B domain has less antibody binding sites and therefore has lower ELISA reading.These results show, in hemophilia A mice, chromogenic assay and ELISA assay can produce the consistent measurement of FVIII expression.Partial thromboplastin time (PTT) has also been carried out at several time points and measures.Result is basically consistent with the result of other two kinds of assay methods.

Example 3

Factor VIII heavy chain mutation

Previous literature has shown that the heavy chain of FVIII is the limiting factor. The heavy chain is processed much less efficiently and/or is more unstable than the light chain, with the precise mechanism remaining unclear. For example, when the heavy and light chain genes are expressed from two separate vectors, a much higher copy number of the heavy chain gene is required than the light chain gene to obtain protein concentrations with similar coagulation activity. Therefore, we set out to improve the efficiency of post-translational processing of the FVIII heavy chain by introducing mutations in its C-terminal region, specifically by introducing new glycosylation sites, as glycosylation is well known for its role in post-translational processing and stability of cell membrane-associated and secreted proteins. We chose to introduce glycosylation at the C-terminus of the heavy chain. The rationale was that, as shown by X-ray crystal structure analysis of the BDDFVIII protein, this terminal region is amorphous and therefore a flexible region without a defined structure. Therefore, mutations in this region are unlikely to disrupt the functional structure of the FVIII protein. Based on this rationale, we utilized the two natural asparagines at positions 734 and 735 and mutated the adjacent amino acids 736 and 737 to threonine (NNAI to NNTT). The mutations minimized the changes in the amino acid sequence and maximized the degree of glycosylation, resulting in two de novo glycosylation sites (NNT and NTT) in the mutant FVIII designated X0 ( FIG8 ).

In addition to the two new glycosylation sites in mutant X0, we added another glycosylation site next to the NNTT site by replacing the peptide (EPRSF) at amino acids 738-742 at the N-terminus of the heavy chain (with the native glycosylation sequence (YVNRSL) separated from amino acids 237 to 242). This native glycosylation site (NRS) was selected because it is one of the most efficient glycosylation sites in the heavy chain (Medzihradszky et al., Anal. Chem. 69: 3986 (1997)), resulting in a mutant FVIII named X1 (Figure 8).

Alternatively, we have replaced amino acids EPR at positions 738-740 with NNT in mutant X0, resulting in two additional glycosylation sites in mutant X2 (Figure 8).

Example 4

Mutations at the C-terminus of the factor VIII heavy chain enhance the in vitro activity of FVIII

Next, we compared the FVIII activity of the mutant constructs with their wild-type BDD FVIII counterparts. Plasmid transfection was performed in the human hepatoma cell line Huh7, which lacks endogenous FVIII expression but is amenable to gene expression controlled by a liver-specific promoter. As shown in Figure 9A, mutations X1 and X2 more than doubled FVIII activity in vitro compared to the parental gene.

Example 5

Long-term and high-level gene expression of mutant FVIII in a hemophilia A mouse model in vivo

Because FVIII mutants X1 and X2 show significantly higher FVIII activity in in vitro transfection experiments, we then examined their gene expression and FVIII activity in FVIII KO mice. The X1 and X2 genes are under the transcriptional control of the same LXP3.3I promoter and are packaged in AAV8 vector particles. Based on early experiments, standard vector doses of 5X 10 ¹⁰ vg/ mice were selected and injected intravenously into hemophilia A mice aged 2 to 3 months via the tail vein. Plasma samples were collected by retroorbital bleeding technique, which is a commonly used method. As shown in Figure 9 B, the expression achieved by mutants X1 and X2 was higher than that of their wild-type parent BDD FVIII gene (low dose compared to Figure 7 A). In order to monitor long-term gene expression, we retained the hemophilia A mice treated with X1 or X2 mutants to observe for 24 weeks. Similar to hemophilia A mice treated with the parental wt BDDFVIII vector (Figure 7A), mice treated with the mutant FVIII vector also showed long-term stable gene expression without significantly reducing human FVIII activity in plasma. Inhibitor testing at different time points showed no inhibitor formation.

Those skilled in the art will appreciate that many and various modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the form of the present invention is only illustrative and is not intended to limit the scope of the present invention.

All publications, patent applications, patents, patent publications, sequences identified by database accession numbers, and other references mentioned herein are incorporated by reference in their entirety for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

The foregoing is illustrative of the present invention and should not be construed as limiting the present invention. The present invention is defined by the following claims, including equivalents of the claims to be included.

sequence:

SEQ ID NO: 1 LXP3.3 promoter-200 bp

SEQ ID NO: 2 (LXP3.3I-288 bp containing LXP3.3 promoter and VH4 intron

SEQ ID NO: 3 Synthetic human B domain deleted factor FVIII coding sequence - 4374 bp

SEQ ID NO: 4 Human factor FVIII gene expression cassette in AAV vector, from left inverted repeat to right inverted terminal repeat - 5045 bp

SEQ ID NO: 5 Human factor FVIII wild-type protein sequence without signal peptide - 2333aa.

Claims (27)

1. A polynucleotide comprising a synthetic liver-specific promoter, wherein the nucleotide sequence of the promoter is SEQ ID NO:1. 2. The polynucleotide of claim 1, wherein the promoter is operatively linked to an intron. 3. The polynucleotide of claim 2, wherein the intron is derived from VH4. 4. The polynucleotide according to claim 3, wherein the nucleotide sequence of the promoter and the intron together is SEQ ID NO:2. 5. The polynucleotide according to any one of claims 1-4, wherein the promoter is operatively linked to the polynucleotide of interest. 6. The polynucleotide of claim 5, wherein the polynucleotide of interest encodes a polypeptide or a functional nucleic acid. 7. The polynucleotide of claim 6, wherein the polynucleotide of interest encodes a coagulation factor. 8. The polynucleotide of claim 7, wherein the polynucleotide of interest encodes factor VIII. 9. The polynucleotide of claim 8, wherein the polynucleotide of interest encodes factor VIII, which is missing a B domain. 10. The polynucleotide of claim 9, wherein the nucleotide sequence of the polynucleotide of interest is SEQ ID NO:3. 11. The polynucleotide according to claim 9, wherein the nucleotide sequence is SEQ ID NO:4. 12. The polynucleotide of claim 5, further comprising a polyadenylation site downstream of the polynucleotide of interest. 13. The polynucleotide of claim 10, further comprising a polyadenylation site downstream of the polynucleotide of interest. 14. The polynucleotide of claim 11, further comprising a polyadenylation site downstream of the polynucleotide of interest. 15. A vector comprising any one of the polynucleotides of claims 1-6 and 12. 16. A vector comprising the polynucleotide of any one of claims 7-11 and 13-14. 17. The vector according to claim 15, wherein the vector is a viral vector. 18. The vector according to claim 16, wherein the vector is a viral vector. 19. The vector according to claim 17, wherein the vector is an adeno-associated virus vector. 20. The vector according to claim 18, wherein the vector is an adeno-associated virus vector. 21. The vector according to claim 19, wherein the adeno-associated virus vector is an AAV8 vector or an AAV9 vector. 22. The vector according to claim 20, wherein the adeno-associated virus vector is an AAV8 vector or an AAV9 vector. 23. A transformed cell comprising a polynucleotide of any one of claims 1-6 and 12 or a vector of any one of claims 15, 17, 19 and 21, wherein the transformed cell does not contain germ cells. 24. A transformed cell comprising any one of the polynucleotides of claims 7-11 or any one of claims 16, 18, 20 and 22, wherein the transformed cell does not comprise germ cells. 25. Use of the polynucleotide of claim 6, the vector of any one of claims 15, 17, 19 and 21, or the transformed cell of claim 23 in the preparation of a medicament for producing polypeptides or functional nucleic acids in the liver of a subject. 26. Use of the polynucleotide of any one of claims 8-11, the vector of any one of claims 16, 18, 20 and 22, or the transformed cell of claim 24 in the preparation of a medicament for treating a subject with hemophilia A or acquired factor VIII deficiency. 27. Use of the polynucleotide of any one of claims 8-11, the vector of any one of claims 16, 18, 20 and 22, or the transformed cell of claim 24 in the preparation of a medicament for increasing the bioavailability of factor VIII polypeptide in a subject.