WO2000024759A1

WO2000024759A1 - Systemic delivery of gene products via skin

Info

Publication number: WO2000024759A1
Application number: PCT/US1999/024773
Authority: WO
Inventors: Haig H. Kazazian, Jr.; Steve Fakharzadeh
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 1998-10-23
Filing date: 1999-10-22
Publication date: 2000-05-04
Also published as: AU1598200A

Abstract

The invention includes compositions and methods for treatment of hemophilia A, comprising introducing nucleic acid encoding Factor VIII into the skin of a mammal for delivery of Factor VIII expressed therefrom into the circulation of the mammal.

Description

SYSTEMIC DELIVERY OF GENE PRODUCTS VIA SKIN

GOVERNMENT SUPPORT This invention was supported in part by funds from the U.S. Government (National Institutes of Health Grant No. HL 38165-13) and the U.S. Government may therefore have certain rights in the invention.

FIELD OF THE INVENTION The field of the invention is the systemic delivery of gene products via the skin in mammals.

BACKGROUND OF THE INVENTION Hemophilia A is an X-linked bleeding disorder that affects approximately 1 to 2 in 10,000 male births. The disorder is caused by defects in the gene encoding the Factor VIII coagulation protein necessary in the blood coagulation cascade process. Patients suffering from hemophilia A develop spontaneous hemorrhaging into joints, muscles, or other internal organs. Unfortunately, due to the short half-life of Factor VIII in the blood stream, treatment of this disorder is mostly limited to infusion therapy administered in response to bleeds or prophylactically before surgery. Further, Factor VIII infusion therapy is extremely expensive and may expose the patient to the risk of infection with human pathogens.

Several factors render hemophilia A an attractive candidate for development of gene replacement therapy. As stated previously, the short half-life of

Factor VIII makes it difficult to sustain adequate systemic levels of the protein despite infusion which is also costly. Nonetheless, even low levels of Factor VIII protein give rise to phenotype correction wherein most patients exhibit clinically significant improvement at 5% of normal protein level. Thus, constant delivery of even small quantities of Factor VIII, such as may be achieved by gene replacement therapy, significantly ameliorates hemophilia A. The epidermis of human skin is an attractive target tissue for gene therapy. The skin, which is the body's largest organ, is capable of producing large quantities of growth factors and cytokines which suggests that it may be a useful bioreactor for the production of therapeutic foreign gene products. Moreover, skin cells may be readily obtained, cultured, and passaged. More importantly, techniques for the introduction of foreign genes into epithelial cells have been developed and tissue culture and grafting techniques are well-established for the grafting of genetically altered skin substitutes. Further, the skin is easily accessible such that the introduction of foreign genes can be easily accomplished, monitored, and controlled so that unwanted events can be easily detected and removed. Human skin is comprised of an outermost epidermis which is separated from the dense connective tissue dermis by a basement membrane zone. The epidermis is further subdivided into a basal compartment made up of proliferating keratinocytes which gives rise to a suprabasal compartment of differentiated keratinocytes. Because the entire vasculature of the skin is within the dermis, all gene products synthesized in the epidermis must traverse the basement membrane to reach the circulation. Several foreign genes have been expressed in epithelial cells and have been shown to traverse the basement membrane (Alexander et al., 1995, Human Mol. Genetics 4:993-999). In addition, several promoters which govern the expression of proteins which are expressed in large quantities in the epidermis (e.g., keratins, involucrin) have been well-characterized and can be used to drive the long-term, tissue-specific expression of desired foreign gene products in skin cells (Vogel, 1993, Arch. Dermatol. 129:1478- 1483). Additionally, tissue culture methods for the in vitro growth of skin substitutes from dissociated keratinocytes and grafting techniques therefor are well-established (Krueger et al., 1994, J. Invest. Dermatol. 103:765-845). Although several recent studies have shown that epithelial cells are capable of expressing foreign genes of interest, sustained and efficient delivery of factors into the bloodstream by keratinocytes expressing a transgene has not been reported (Vogel, 1993, Arch. Dermatol. 129:1478-1483). The expression of several human proteins not normally expressed in biologically significant levels in keratinocytes has been achieved with subsequent detection of the gene products in the blood plasma. However, all of these factors have comprised relatively small proteins requiring little or no post-translational modification. Human Factor VIII undergoes extensive intracellular post-translational modification through N-glycosylation of asparagine residues and sulfonation of tyrosine residues. Factor VIII is synthesized as a large (265 kDa) precursor which must be cleaved to generate a heavy chain and a light chain (Figure 1). The chains then associate as a heterodimer which circulates as an inactive precursor protein stabilized by association with von Willebrand's factor until it is activated by cleavage by thrombin (factor Ila). Thus, the expression and secretion of biologically active Factor VIII in a cutaneous transgenic delivery system poses technical difficulties not yet addressed by the limited success of the prior art.

The introduction and expression of foreign genes into human keratinocytes through transduction with retroviral vectors has been described for human growth hormone (HGH) which is normally expressed in the hypothalamus (Morgan et al., U.S. Pat. No. 4,980,286). However, HGH is a relatively small protein

(29 kDa) which requires little or no post-translational modification.

In one study human keratinocytes were transfected with a recombinant plasmid vector such that they expressed a mutant human apolipoprotein E (apoE) containing an influenza virus hemagglutinin epitope (HA1), i.e., the 284 amino acid human apoE polypeptide contained the HA1 epitope at amino acid positions 274-283

(Fenjves et al., 1994, Human Gene Therapy 5:1241-1248). The keratinocytes, which expressed the human apoE-HAl fusion protein, were grafted to athymic mice and the human apoE-HAl fusion protein was detected in the sera of several recipient athymic mice. Unlike Factor VIII, apoE is normally secreted by keratinocytes and is a small (34 kDa) protein which requires little or no post-translational modification. Therefore, it was anticipated that apoE would be expressed in and secreted from keratinocytes into the circulating blood.

Secretion of human factor IX, a coagulation cascade protein which is defective in hemophilia B patients, into the bloodstream of nude mice following transplantation of transduced human keratinocytes was reported by Greenhalgh et al. (1994, J. Invest. Dermatol. 103:63S-69S). However, the authors noted that "long term expression was not sustained in the circulation" (id. at 67S). Factor IX is a small 56 kDa protein and therefore, it is expected that the protein would readily cross the basement membrane zone.

More recently, transgenic mice were shown to express and secrete human Factor IX in the skin but not in other tissues where cytokeratin gene promoters were used to drive expression (Alexander et al., 1995, Human Mol. Gen. 4:993-999). The tissue-specificity of this study is important since normally Factor IX is expressed by hepatocytes, and specific targeting of gene expression to the skin is an important feature of a cutaneous gene delivery system. While this study demonstrates the skin- specific expression of Factor IX in transgenic mice by keratinocytes and demonstrates that a small protein expressed by suprabasal keratinocytes can be secreted into the systemic circulation, successful expression of Factor IX does not present the more difficult challenges of expression of the much larger and more complex Factor VIII.

There is a present and acute need for a delivery method which provides functional circulating Factor VIII to hemophilia A patients. Even low levels of circulating Factor VIII would provide significant therapeutic relief to such patients. The present invention satisfies this need.

SUMMARY OF THE INVENTION The invention includes a composition comprising an isolated human Factor VIII nucleic acid, or a biologically active fragment thereof, wherein the Factor VIII nucleic acid has a first mutation comprising a deletion of a portion of the "B" domain of the Factor VIII nucleic acid, the deletion effecting the removal of both cleavage sites of the heavy and light chains and a part of the acidic 2 domain of the human Factor VIII protein.

The invention also includes a composition comprising an isolated human Factor VIII nucleic acid, or a biologically active fragment thereof, wherein a portion of the "B" domain of the human Factor VIII is deleted, the human Factor VIII nucleic acid further comprising a second mutation, wherein the second mutation reduces the ability of the human Factor VIII protein to bind a chaperone protein. In one aspect, the chaperone protein is immunoglobulin Binding Protein/glucose-regulated protein of 78 kDa ("BiP/GRP78").

In another aspect, the second mutation effects a non-conservative amino acid substitution at amino acids 291 through 309 of the human Factor VIII protein.

In yet another aspect, the second mutation substitutes a phenylalanine amino acid residue with a serine amino acid residue at position 309 in the mature human Factor VIII protein.

In another aspect, the nucleic acid further comprises a promoter/regulatory sequence operably linked to the human Factor VIII nucleic acid, wherein the promoter/regulatory sequence is positioned at the 5' end of the human Factor VIII nucleic acid. In a preferred embodiment, the promoter/regulatory sequence is selected from the group consisting of an inducible promoter, a constitutive promoter and a tissue specific promoter, a basal skin layer specific promoter, a suprabasal skin layer promoter, a keratin promoter, an involucrin promoter, a cytokine promoter, a growth factor promoter, the SV40 early promoter, a retroviral LTR promoter and the cytomegalovirus immediate early promoter.

In another aspect, the nucleic acid further comprises a second regulatory sequence positioned between the 3' end of the promoter/regulatory sequence and the 5' end of the human Factor VIII nucleic acid.

In a preferred embodiment, the second regulatory sequence is an intron. In a more preferred embodiment, the intron is selected from the group consisting of an SV40 intron, and a rabbit β-globin intron.

In another aspect, the nucleic acid comprises a polyadenylation signal sequence positioned at the 3' end of the human Factor VIII nucleic acid. In a preferred embodiment, the polyadenylation signal sequence is selected from the group consisting of an SV40 polyadenylation signal sequence, a rabbit β-globin polyadenylation signal sequence and a keratin polyadenylation signal sequence.

In yet another preferred embodiment, the promoter/regulatory sequence is the involucrin promoter, and the second regulatory sequence is the SV40 intron, and further, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence.

In another preferred embodiment, the promoter/regulatory sequence is a keratin promoter, and the second regulatory sequence is the rabbit β-globin intron, and further, the polyadenylation signal sequence is a keratin polyadenylation signal sequence.

In yet another preferred embodiment, the promoter/regulatory sequence is a keratin promoter, and the second regulatory sequence is the SV40 intron, and further, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence.

Also included in the invention is a composition comprising an isolated human Factor VIII protein, or a biologically active fragment thereof, wherein the protein comprises a first mutation comprising deletion of amino acids 741 through 1666 of the full-length human Factor VIII protein. In addition, the invention includes a composition comprising an isolated human Factor VIII protein, or a biologically active fragment thereof, wherein a portion of the "B" domain of the human Factor VIII is deleted, the human Factor VIII protein further comprising a second mutation which reduces the ability of the human Factor VIII to bind a chaperone protein. In one aspect, the chaperone protein is BiP/GRP78.

In another aspect, the second mutation effects a non-conservative amino acid substitution at amino acids 291 through 309 in the human Factor VIII protein.

In yet another aspect, the second mutation causes the substitution of a phenylalanine amino acid residue with a serine amino acid residue at position 309 in the human Factor VIII protein.

Further included in the invention is a cell comprising an isolated human Factor VIII nucleic acid, or a biologically active fragment thereof, wherein the Factor VIII nucleic acid has a first mutation comprising a deletion of a portion of the "B" domain of the Factor VIII nucleic acid, the deletion effecting the removal of both cleavage sites of the heavy and light chains and a part of the acidic 2 domain of the human Factor VIII protein.

In one aspect, the cell is a mammalian cell.

In a preferred embodiment, the cell is selected from the group consisting of a mouse, a rat and a human cell.

In an more preferred embodiment, the cell is an epidermal cell.

In an even more preferred embodiment, the cell is selected from the group consisting of suprabasal keratinocytes and basal keratinocytes.

In yet another preferred embodiment, the cell is a human cell. The invention additionally includes a vector comprising an isolated

Factor VIII nucleic acid, or a biologically active fragment thereof, wherein the Factor VIII nucleic acid has a first mutation comprising a deletion of a portion of the "B" domain of the Factor VIII nucleic acid, the deletion effecting the removal of both cleavage sites of the heavy and light chains and apart of the acidic 2 domain of the human Factor VIII protein, or a modification or portion thereof.

The invention further includes a nonhuman transgenic mammal encoding the human Factor VIII nucleic acid of claim 1, the nucleic acid further comprising a promoter/regulatory sequence operably linked to the human Factor VIII nucleic acid, and further wherein the human Factor VIII nucleic acid is expressed in the transgenic animal, and wherein the transgenic mammal exhibits a phenotypic correction compared with an otherwise identical nontransgenic littermate. In one aspect, the mammal is a mouse.

In a preferred embodiment, the phenotypic correction is the shortening of coagulation time of the blood of the mammal compared with the coagulation time of the blood of an otherwise identical nontransgenic littermate.

Also included in the invention is a method of expressing an isolated human Factor VIII nucleic acid in the skin of a mammal wherein the Factor VIII is secreted into the circulation of the mammal. The method comprises introducing the isolated nucleic acid into skin cells, culturing the skin cells in vitro, and grafting the skin cells onto the mammal, wherein the isolated human Factor VIII nucleic acid has a first mutation comprising a deletion of a portion of the "B" domain of the Factor VIII nucleic acid, the deletion effecting the removal of both cleavage sites of the heavy and light chains and a part of the acidic 2 domain of the human Factor VIII protein In addition, the invention includes a method of delivering Factor VIII to the blood of a mammal. The method comprises administering to the skin of the mammal nucleic acid encoding Factor VIII.

The invention further includes a method of treating hemophilia A in a mammal. The method comprises obtaining skin cells from a mammal, introducing an isolated human Factor VIII nucleic acid into the cells, culturing the skin cells in vitro, and grafting the skin cells expressing Factor VIII onto the mammal, wherein the Factor VIII nucleic acid has a first mutation comprising a deletion of a portion of the "B" domain of the Factor VIII nucleic acid, the deletion effecting the removal of both cleavage sites of the heavy and light chains and a part of the acidic 2 domain of the human Factor VIII protein.

Further included is a method of treating hemophilia A in a mammal. The method comprises obtaining skin cells from a mammal, introducing an isolated human Factor VIII wild type nucleic acid into the cells, culturing the skin cells in vitro, and grafting the skin cells expressing Factor VIII onto the mammal. BRIEF DESCRIPTION OF THE.DRAWINGS Figure 1 is a diagram depicting the domain structure and proteolytic cleavage sites of human Factor VIII. Human Factor VIII is expressed initially as a large 265 kDa precursor protein of the domain structure shown in the top of the diagram. Next, the precursor is cleaved within the "B" domain to generate a heavy polypeptide chain of 200 kDa and a light polypeptide chain of 80 kDa. Both heavy and light chains must associate with von Willebrand's factor to reconstitute functional Factor- VIII. Factor VIII is activated by Factor Ila (thrombin) cleavage.

Figure 2 A is a diagram depicting the structure of full-length Factor VIII.

Figure 2B is a diagram depicting the structure of the "B" domain- deleted variant of human Factor VIII, termed VEIL A. In this variant, amino acids 741- 1639 have been deleted to shorten the cDNA encoding this polypeptide to approximately 4.7 kb and to reduce the precursor protein to approximately 165 kDa. The "B" domain-deleted precursor undergoes proteolytic cleavage to generate a truncated heavy chain and a normal light chain.

Figure 3 is a diagram depicting the pinvVIIILA transgene construct. From 5' to 3', the construct contains the following: involucrin promoter region (including the 1.2 kb intron critical for tissue-specific expression); SN40 intron sequences; human Factor VEIL A ("B" domain-deleted variant) cDΝA; and SV40 polyadenylation signal. The transgene is liberated from the vector backbone by Sail digestion. The 5' and 3' primers for RT-PCR analysis flank the intron sequences as shown. The transcription initiation site is designated by the arrow.

Figure 4 is a diagram illustrating the mouse mating strategy employed to obtain transgenic mice in a FVIII null or deficient background. Νon-transgenic, female mice homozygous for the Factor VIII knockout allele (X^"X7TT^") were mated with male transgenic mice (X⁺Y/TT). All FI male mice (X^"Y) inherit the Factor VIII null allele from their mothers. Of these FI males, one-half would be predicted to inherit the human Factor VIII transgene from the father ( T) assuming integration of the transgene sequences into autosomal chromosomes. These transgenic mice (X' Y/TT^") should express only transgenic human Factor VIII in the epidermis but not express any normal murine Factor VIII. Figure 5 is an image of a DNA gel demonstrating the expression of the pinvVIIILA transgene in various tissues obtained from a mouse from pinvVIIILA transgenic mouse using RT-PCR analysis. The primer pair depicted in Figure 3 was used to detect a pinvVIIILA message-specific amplification product of 280 bp. From left to -right, the lanes were loaded with RT-PCR products from the following tissues: skin, brain, tongue, esophagus, thymus, lung, heart, liver, intestine, colon, kidney, bladder, testes, and a lane showing various size markers.

Figure 6 is a diagram illustrating the strategy for the generation of Factor VIELAΔ cDNA. The diagram demonstrates a schematic of Factor VEIL A "B" domain-deleted protein showing the three "A" domains, two "C" domains, the two acidic regions, and the small remaining portion of the "B" domain shown in white.

Immediately below the Factor VEIL A protein schematic, is an illustration of the VIIILA cDNA sequence within the region targeted for deletion. The upstream primer pair indicated by facing arrows was designed to amplify the sequences between codon 740 and the unique 5' Kpnl site. The downstream primer pair was designed to amplify sequences between codon 1667 and the unique 3' Apal site. Pfu polymerase was used for PCR amplifications to generate fragments with blunt ends. The upstream and downstream fragments were digested with Kpnl and Apal, respectively. A three-way ligation into Kpnl-Apal-digested Factor VIIILA cDNA resulted in juxtaposition of codons 740 and 1667 as shown in the bottom schematic thus extending the "B" domain deletion by the desired 81 base pairs.

Figure 7 is an image of a DNA gel depicting genotype analysis of Factor VIE/RAG 1 double knockout mice. Polymerase chain reaction primer pairs specific for the Factor VIII knockout allele (FVIII KO), the Factor VIII wild type allele (FVIII WT), the RAG1 knockout allele (RAG1 KO), and the RAG1 wild type allele (RAG1 WT) were used for genotype analysis of F2 mice derived from Factor VIII knockout mouse matings with RAG1 knockout mice. .The genotype results for a female double knockout mouse (R^"R^"X"X") and a female wild type mouse (R⁺R⁺X⁺X⁺) are shown in the top panel. Genotype results for a male double knockout mouse (R^"R^" X^"Y) and a male wild type mouse (R⁺R⁺X⁺Y) are shown in the lower panel. Double knockout mice show only amplification products for the knockout alleles, whereas wild type mice show only amplification products specific for the wild type alleles.

Figure 8 is a diagram illustrating the strategy for introducing a point mutation which effects a change at amino acid position 309 of the human Factor VIII B-domain protein from a phenylalanine to a serine. A schematic of the Factor VIIILA protein is shown at the top of the diagram. The upper nucleic acid sequence represents the normal Factor VIII DNA sequence in the region of Phe³⁰⁹. Adjacent primers oriented in opposite directions were designed as shown. The upstream primer has a "T" to "C" mutation at codon 309, thus converting the amino acid at this position from phenylalanine to serine. The upstream primer pair amplifies the region between codon

315 and a unique 5' Spel site. The downstream primer pair amplifies a region between codon 316 and a unique 3' Kpn I site. PCR amplification is performed using Pfu polymerase to generate blunt-end amplification products. After digestion of the upstream and downstream amplification products with Spe I or Kpn I, respectively, a three-way ligation into Spe I-Kpn I-digested Factor VIIILA cDNA is performed.

Ligation of the blunt ends of the PCR-amplified fragments juxtaposes codons 315 and 316, thus reconstituting the appropriate reading frame while incorporating the phenylalanine to serine mutation at codon 309.

DETAILED DESCRIPTION OF THE INVENTION The invention is based on the discovery that expression of nucleic acid encoding human Factor VIII in the epidermis of a mammal results in delivery of the Factor VIII protein from the skin to the systemic circulation. The protein is thus present in the plasma of the mammal at therapeutic levels as demonstrated by the phenotypic correction exhibited by the mammal. Therefore, the invention is of tremendous therapeutic benefit to a mammal having hemophilia A.

The invention relates to a method for the expression of human Factor VIII, or a biologically active portion or variant thereof, in a transgenic animal wherein the Factor VIII is secreted into the systemic circulation at therapeutic levels which provide phenotypic correction of a hemophilia A phenotype in the transgenic mammal.

The invention further relates to a transgenic animal having Factor VIE nucleic acid contained therein, as described in the experimental examples presented herek r The invention also relates to a method for treatment of hemophilia A in a mammal wherein epidermal cells expressing a foreign Factor VIII cDNA, or a portion or variant thereof encoding a biologically active portion of Factor VIII, are grafted onto a mammal thereby inducing therapeutic levels of Factor VIII protein in the systemic circulation of the mammal. The Factor VIII cDNA which is introduced into the epidermal cells is either wild type Factor VIII or is a mutant or variant thereof, as described in detail herein.

By "treatment," as the term is used herein, is meant any therapy rendered to a mammal, preferably a human, for the purpose of preventing, alleviating, or ablating a blood protein deficiency disease, whether or not clinical symptoms of the disease are present in the mammal.

By "therapeutic effect," as the term is used herein, is meant any decrease in the blood clotting time of an animal which has received a treatment compared with the blood clotting time of the animal before the treatment and/or with the blood clotting time of an otherwise identical animal which has not received the treatment. The invention is not limited solely to the production of transgenic animals which express Factor VIII in their skin tissues. Rather, the invention should be construed to include delivery of other blood coagulation factors the deficiency in which causes a disease or condition in a mammal, using any method of gene delivery to the epidermis. Thus, the invention should be construed to include: delivery of Factor VII for treatment of Factor VII deficiency (Petersen et al., 1995, "Factor VII" In: Molecular Basis of Thrombosis and Hemostasis. High and Roberts, eds., Marcel Dekker, Inc.); delivery of Factor IX to a mammal for treatment of hemophilia B (High et al., 1995, "Factor IX", ibid.); delivery of Factor X for treatment of Factor X deficiency (Watzke et al., 1995, "Factor X", ibid); delivery of Factor XI for treatment of Factor XI deficiency (Fujikawa et al., 1995, "Factor XI", ibid.); delivery of Factor XIII for treatment of Factor XIII deficiency (Lai et al., 1995, "Factor XIII", ibid.); and delivery of Protein C for treatment of Protein C deficiency (Suzuki, 1995, "Protein C", ibid.). By "blood protein deficiency disease," as the term is used herein, is meant any disease state which may be ameliorated by supplying the protein to the systemic circulation of an animal.

By "blood coagulation disease," as the term is used herein, is meant any disease state characterized by an increased bleeding time in a coagulation assay performed as described herein. In a preferred embodiment, the protein of interest is delivered to the systemic circulation of a mammal by creating a transgenic animal wherein an isolated nucleic acid encoding Factor VIII is operatively linked to the involucrin promoter/regulatory region such that the Factor VIII encoded thereby is selectively expressed mainly in the epidermal cells of the transgenic mammal. However, the present invention is not limited solely to expression in transgenic animals. Rather, the invention encompasses delivery of a protein to the systemic circulation of a mammal wherein epidermal cells expressing the gene and the protein of interest are grafted onto a recipient animal. The invention encompasses transplant of skin cells from a transgenic donor mammal onto a recipient animal. Additionally, the invention includes the in vitro culturing of skin cells obtained from an animal, introducing an isolated nucleic acid encoding a protein of interest into the cells, and grafting the cells expressing the nucleic acid back onto either the original donor animal or another recipient animal, all according to standard methods (see, e.g., Anderson et al., 1995, U.S. Pat. No. 5,399,346; Fenjves et al, 1994, Human Gene Ther. 5:1241-1248; Greenhalgh et al., 1994, J. Invest. Dermatol. 103:63S-69S; Krueger et al., 1994, J. Invest. Dermatol.

Skin grafts may be performed to deliver Factor VIII to the epidermal tissue as described in detail herein. Therefore, the invention is not limited to the introduction of isolated nucleic acid encoding Factor VIII into mammalian oocytes by micro injection. Instead, the invention includes the introduction of exogenous nucleic acids into epidermal cells using a variety of methods including, but not limited to, viral vector -mediated gene transfer, for example, retroviral-mediated gene transfer, electroporation, calcium phosphate-mediated transfection, plasmid-based vectors, microinjection, liposomes, and the like. Also, delivery of each of the above-recited proteins to the cells of a mammal may be accomplished by an ex vivo approach by first harvesting epidermal cells, introducing an exogenous DNA encoding a protein of interest which is expressed in the cells which would not otherwise express the protein in biologically significant amounts, culturing the cells in vitro, and then grafting the exogenous DNA-containing cells onto a mammal. Proteins are therefore delivered by way of expression of the exogenous DNA. Alternatively, isolated nucleic acid encoding Factor VIII may be administered directly to the skin of a mammal by procedures such as liposomal (i.e., a "gene cream" approach) and/or ballistic gene transfer techniques (Greenhalgh et al., 1994, J. Invest. Dermatol. 103:63S-69S). Direct injection into the epidermal layer of the skin is also contemplated in the invention.

The nucleic acid encoding Factor VIII to be administered to the mammal may be combined with other nucleic acid sequence elements to form a "Factor VIII construct". A Factor VIII construct is useful for effecting enhanced expression of Factor VIII in the cells into which the Factor VIII construct is introduced. In addition, depending on the nature of the Factor VIII construct, it is possible to deliver the nucleic acid encoding Factor VIII to the mammal wherein the gene is expressed in a tissue specific manner or in an inducible manner, both of which methods of expression may enhance expression of Factor VIII where and when it is required, while minimizing the expression of Factor VIII in tissue locations wherein Factor VIII expression is not desired. Thus, the nucleic acid encoding Factor VIII may be combined with other nucleic acid sequences that encode regulatory elements, including but not limited to, promoter/regulatory sequences, transcription enhancers and transcription terminators. Promoter/regulatory sequences that may be used in conjunction with this invention include promoters used alone or in conjunction with other DNA sequence elements such as enhancers, or elements that confer inducible or tissue-specific expression on the nucleic acid encoding Factor VIII. By way of example, skin-specific promoters such as the involucrin promoter, or promoters which are derived from genes encoding proteins that are highly expressed in keratinocytes, may be used.

The invention, however, should not be interpreted as being limited to the presence of any or all of these particular elements or to any particular arrangement thereof. Rather, the invention encompasses other promoter/regulatory regions, enhancers, polyadenylation signal sequences, and the like, arranged in various orders and permutations thereof. Further, the invention includes constructs which do not have one or more of the above-stated DNA elements.

An "isolated nucleic acid", as used herein, refers to a nucleic acid sequence, segment, or fragment which has been separated from the sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g. , the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

The term "isolated nucleic acid sequence" includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or any modified forms thereof, including chemical modifications of the DNA or RNA for enhanced stability which render the nucleic acid more stable when it is either cell free or when it is associated with a cell. Chemical modifications of nucleic acid may also be used to enhance the efficiency with which a nucleic acid is taken up by a cell or the efficiency with which it is expressed in a cell. -Any and all combinations of modifications of the nucleic acids are contemplated in the present invention.

The isolated nucleic acid sequence may be derived from a biological source or it may be derived by chemical synthesis. Examples of a nucleic acid encoding Factor VIII that may be used in the present invention is the human Factor VIII as set forth in (Wood et al., 1984, Nature 312:330-337; Tool et al., 1986, Proc. Natl. Acad. Sci. 83:5939-5942). Additionally, isolated nucleic acids encoding Factor VIII derived from other mammals may also be used in accordance with this invention provided they are homologous to the human Factor VIII described herein and they encode a Factor VIII protein, or a portion thereof, which has Factor VIII activity as defined herein. The present invention should be construed to include any and all homologous nucleic acids encoding Factor VIII and Factor VIII proteins having Factor

VIII activity as defined herein.

The term "homologous", as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3' ATTGCC 5' and 3' TATGCG 5' share 50% homology. Preferably, the nucleic acid encoding Factor VIII useful in the methods of the present invention is at least about 50% homologous to the human Factor VIII disclosed in the following references (Wood et al, 1984, Nature 312:330-337; Tool et al., 1986, Proc. Natl. Acad. Sci. 83:5939-5942; Gitschier et al., 1984, 312:326-330). More preferably, the Factor VIII is about 65% homologous, preferably it is about 75% homologous and even more preferably, the factor useful in the methods of the invention is about 90 to about 95% homologous to the Factor VIII isolated nucleic acid sequence disclosed in the following references (Wood et al., 1984, Nature 312:330- 337; Tool et al., 1986, Proc. Natl. Acad. Sci. 83:5939-5942; Gitschier et al, 1984, 312:326-330). The term "nucleic acid encoding Factor VIE", as used herein, is also meant to include an isolated nucleic acid sequence encoding at least a portion of a mammalian Factor VIII protein having Factor VIII activity, or variants thereof. Such variants, i.e., analogs of proteins or polypeptides of human Factor VIII, include proteins or polypeptides which have been or may be modified using recombinant DNA technology such that the protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to, variants conferring enhanced stability on the protein in plasma, enhanced ability to be secreted into the systemic circulation, and enhanced specific activity of the protein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; . aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. Non-conservative amino acid substitutions, on the other hand, are those outside the above-Jisted groups. In the present invention, the non-conservative substitution is the replacement of a serine amino acid in place of a phenylalanine amino acid.

Preferably, the amino acid sequence of an human Factor VIE analog is about 70% homologous, more preferably about 80% homologous, even more preferably about 90% homologous, more preferably, about 95% homologous, and most preferably, at least about 99%) homologous to the amino acid sequence of human Factor NIII described in the following references (Wood et al., 1984, Nature 312:330-337;

Tool et al, 1986, Proc. Natl. Acad. Sci. 83:5939-5942; Gitschier et al., 1984, 312:326- 330).

Any number of procedures may be used for the generation of mutant or variant forms of human Factor VIII. For example, generation of mutant forms of human Factor VIII which do not bind the BiP/GRP78K chaperone protein may be accomplished by introducing deletion, substitution or insertion mutations into a human nucleic acid encoding Factor VIII residing on a plasmid template using ordinary recombinant DNA methodology described in any molecular biology manual, for example, described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY). DNA encoding a mutated human Factor VIII protein which does not bind BiP/GRP78K but retains human Factor VIII biological activity, is suitable for use in the vector of the invention. Procedures for the introduction of amino acid changes in a protein or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in Sambrook et al. (1989, supra).

Typically, the nucleic acid encoding Factor VIII will encode a portion of a Factor VIII polypeptide that is at least about fifteen contiguous amino acids, typically at least about twenty five contiguous amino acids, more typically at least about forty contiguous amino acids, usually at least about forty five contiguous amino acids and preferably at least about fifty contiguous amino acids in length. Any length of Factor VIII polypeptide is contemplated in the method of the invention provided the Factor VIII polypeptide used has Factor VIII activity as defined herein.

As used herein, the term "promoter/regulatory sequence" means a DNA sequence which is required for expression of a gene operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene in an inducible/repressible or tissue-specific manner.

By describing two DNAs as being "operably linked" as used herein, is meant that a single-stranded or double-stranded DNA comprises each of the two DNAs and that the two DNAs are arranged within the DNA in such a manner that at least one of the DNA sequences is able to exert a physiological effect by which it is characterized upon the other.

The use of the term "DNA encoding" should be construed to include the DNA sequence which encodes the desired protein and any necessary 5 ' or 3' untranslated regions accompanying the actual coding sequence. The term "expression of a nucleic acid" as used herein means the synthesis of the protein product encoded by the nucleic acid.

By the term "positioned at the 5' end" as used herein, is meant that the promoter/regulatory sequence is covalently bound to the 5' end of the gene whose expression it regulates, at a position sufficiently close to the 5' start site of transcription of the gene so as to drive expression of the gene.

A first region of an oligonucleotide "flanks" a second region of the oligonucleotide if the two regions are adjacent one another or if the two regions are separated by no more than about 1000 nucleotide residues, and preferably no more than about 100 nucleotide residues.

Further, due to the accessibility of the skin, promoters which may be activated by a variety of skin-specific stimuli including, but not limited to, wounding, tape stripping, and topical retinoid application, topical steroids, and the like, may also be useful in inducing skin-specific expression of the nucleic acid encoding Factor VIII construct.

In addition, many promoter sequences are now commercially available. Promoters which are useful include those which are inducible, which are tissue specific and which are constitutive. In preferred embodiments, the promoters which are useful include those which are expressed in the basal and suprabasal layers of the skin. Such promoters include those which are tissue specific, but also include those which drive high level constitutive expression of genes operably linked thereto. Such strong promoters include, for example, a retroviral LTR promoter, the cytomegalovirus immediate early promoter and the SV40 early promoter. A preferred promoter for use in the invention is the involucrin promoter as described in further detail herein. Other suitable promoters include a keratin promoter, a cytokine promoter and a growth factor promoter.

Thus, one of skill in the art will know how to generate a nucleic acid encoding Factor VIII construct having a desired promoter sequence, wherein specific sequences having promoter activity are documented, or by simply purchasing the desired promoter sequence from a commercial source. The actual generation of the Factor VIII construct having the desired promoter sequence is accomplished using ordinary molecular biology technology such as that described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, NY) and Ausubel et al. (1997, Current Protocols in Molecular Biology. Green & Wiley, NY).

Additionally, the nucleic acid encoding Factor VIII may be combined with a viral vector nucleic acid or a plasmid vector nucleic acid which facilitate more efficient introduction of the nucleic acid encoding Factor VIII into cells. Methods of combining nucleic acid sequences are well known to those of skill in the art and are found, for example in Sambrook, et al. (1989, supra).

Vectors derived from viruses that may be used in conjunction with invention include, by way of example, retroviruses, lentiviruses, adenoviruses, adeno- associated viruses, papilloma viruses, and herpesviruses. Other viral vectors and plasmid vectors that may be used in conjunction with this invention are well known to those of skill in the art and are found, for example in Sambrook, et al. (1989, supra) and Ausubel et al. (1997, supra). Use of viral and non-viral vectors is described, for example, in Wilson (1994, Nature 365:691-692). Viral vectors which are useful for delivery of Factor VIII to a mammal may include a virus, which may be a DNA- containing or an RNA-containing virus, which is capable of having inserted into the viral genome a heterologous nucleic acid fused to a promoter sequence for expression thereof. The promoter may be any of the promoter sequences described herein. The viral vector is administered to the mammal, wherein expression in the cells of mammal of Factor VIII results in a decrease of the blood coagulation time in the animal compared with an otherwise identical animal which does not express Factor VIII.

As used herein, the term "viral vector" means a virus particle capable of transducing an animal host cell and into which a desired nucleic acid may be inserted which is expressed in the cells. Thus, the nucleic acid encoding Factor VIII may be formulated for introduction into cells in a mammal in any number of ways. For example, the nucleic acid encoding Factor VIII may be formulated in viral vector, such as a retroviral vector or an adenoviral vector. The nucleic acid encoding Factor VIII may be formulated in liposomes. The nucleic acid encoding Factor VIII may be formulated with various targeting molecules including, but not limited to, targeting ligands, targeting antibodies, or ligands for receptor-mediated DNA uptake.

Examples of other formulations of the nucleic acid encoding Factor VIII that may be used include the following. The nucleic acid encoding Factor VIII may be formulated with cationic lipids for transfer into cells (Fasbender et al, 1995, Am. J. Physiol. 269:L45-L51). The nucleic acid encoding Factor VIII might be formulated with polylysines (poly-L-lysine) containing or complexed with ligands having receptors on target cell types (Curiel et al, 1991, Proc. Natl. Acad. Sci. USA 88:8850- 8854).-The nucleic acid encoding Factor VIII may be formulated with polylysines containing or complexed with sugar residues to facilitate transformation or transfection via membrane lectins (Midoux et al, 1993, Nucleic Acids Res. 21 :871-878; Erbacher et al, 1995, Bioconj. Chem. 6:401-410; Erbacher et al, 1996, Hum. Gene Ther. 7:721- 729).

The nucleic acid encoding Factor VIII may also be formulated using an ex vivo approach wherein the nucleic acid encoding Factor VIII is introduced into keratinocytes in culture, and the cells expressing the nucleic acid encoding Factor VIII are then grafted onto a recipient animal. To accomplish this, keratinocytes are cultured in vivo from a small biopsy and passaged until sufficient cells are obtained. The cells are then transfected with a nucleic acid encoding Factor VIII, such as for example, by transduction of the keratinocytes with a retroviral vector containing the nucleic acid encoding Factor VIII. The transfected keratinocytes containing the nucleic acid encoding Factor VIII are then grafted back either onto the original donor animal or another recipient animal. The number of cells used per graft and the surface area of the graft will depend on the animal being treated and the level of expression of Factor VIII protein achieved in the cells.

By "biologically active Factor VI " as used herein, is meant a mammalian Factor VIII protein, or a portion or variant thereof, which is capable of mediating the coagulation of blood in a blood coagulation assay. Blood coagulation assays are well known in the art and are described, for example, in Walter et al. (1996, Proc. Natl Acad. Sci. USA 93:3056-3061) and in Hathaway and Goodnight (1993, "Laboratory Measurement of Hemostasis and Thrombosis," In: Disorders of Hemostasis and Thrombosis: A Clinical Guide, pp.21-29).

The term "Factor VIII activity," as used herein, defines a protein having at least about 75% homology with the human Factor VIII amino acid sequence of the present invention, which mediates the clotting of blood as determined, for example, by the assays provided herein in the experimental details section. Factor VIII activity may be assayed in vitro or in vivo. In an example of an in vitro assay, Factor VIII- dependent activation of factor X may be measured in a photometric fashion in vitro via the COAMATIC® assay described herein. In an example of an in vivo assay, the mouse model of hemophilia described in the examples is used to assess whether introduction of the nucleic acid encoding Factor VIII into the animal has a therapeutic effect on the status of the hemophilia phenotype of the mammal being treated. By "therapeutic effect" is meant any decrease in the blood clotting time of the mammal compared with the blood clotting time of an otherwise identical animal into which the nucleic acid encoding Factor VIII has not been introduced.

For the purposes of clarity and for the purposes of satisfying the best mode requirement, the discussion which follows exemplifies Factor VIII as the preferred protein to be delivered to the epidermis of a mammal The invention is based on the discovery that expression of human nucleic acid encoding Factor VIII in the skin of a transgenic mammal results in expression of levels of human Factor VIII protein in the blood plasma of the mammal which provides phenotype correction, thereby providing a therapeutic benefit to the mammal. The invention is further based on the fact that human Factor VIII protein is expressed as a large (265 kDa) precursor polypeptide which is later enzymatically cleaved into a heavy (200 kDa) and a light (80 kDa) protein chain. The terms "heavy" and "light" chain refer, therefore, to the two proteins resulting from cleavage of the 265 kDa precursor having molecular weights of about 200 kDa and about 80 kDa, respectively. The heavy and light chain circulate in the blood as a dimer associated with von Willenbrand's factor until it is activated by. cleavage by thrombin (factor Ea) which cleaves the heavy chain into two polypeptides having approximate molecular weights of 50 kDa and 43 kDa and also removes amino acids 1648 through 1688 from the light chain.

In a particularly preferred embodiment, a variant of human Factor VIII cDNA (termed "VIIILA cDNA") is operably linked to the involucrin promoter to drive expression of the transgene in the skin of transgenic mice. However, the invention should- not be construed to be limited to the expression of VIIILA cDNA. Rather, the invention encompasses any nucleic acid encoding Factor VIII having Factor VIII activity substantially similar to the Factor VIII activity described herein.

In a preferred embodiment, the Factor VIII cDNA is further shortened by deleting the nucleotide basepairs encoding amino acids from about amino acid 741 through about amino acid 1666 such that the precursor protein encoded by the shortened cDNA has a molecular weight of approximately 165 kDa compared with the full-size, wild type precursor protein of about 265 kDa. However, the present invention should in no way be construed as being limited to the expression of any particular variant of human Factor VIII. Rather, the invention encompasses expression of any full-length or shorter Factor VIE protein having Factor VIII activity. By "smaller precursor" is meant a precursor protein having at least one fewer amino acid than the full-length 265 kDa precursor protein, which in humans is comprised of amino acids 1 through 2332. Preferably, amino acids 741 through 1666 have been deleted to form the smaller human precursor Factor VIII of the present invention but the invention is not limited thereto. Modifications to generate a smaller precursor polypeptide which is not cleaved into heavy and light chains but which, instead, enters the circulation as a single polypeptide, may enhance the ability of the now smaller precursor to be secreted through the basement membrane zone and may render the protein more stable in the circulation. Further, mutation of amino acid 309 from Phe to Ser reduces the level of binding and sequestration of Factor VIII protein by the chaperone protein, immunoglobulin Binding Protein/glucose-regulated protein of 78 kDa ("BiP/GRP78K") (Swaroop et al, 1997, J. Biol Chem. 272:24121-24124). Decreased binding by BiP/GRP78K may increase circulating levels of Factor VIII. Thus, the delivery of variant forms of human Factor VIII from the skin of a mammal into the systemic circulation may be enhanced by decreased binding of the variant Factor VIII protein to BiP/GRP78K.

The term "truncated heavy chain" refers to a heavy chain polypeptide having at least one fewer amino acid than the normal, full-length, untruncated heavy chain polypeptide which in humans has a molecular weight of about 200 kDa and is comprised of amino acids 1 through 1313. The truncated polypeptide may have fewer amino acids at either the amino or carboxy terminus or both. In a particularly preferred embodiment, amino acids 741 through 1313 of the full-length human Factor VIII protein have been removed from the NH₂ terminus to form a truncated heavy chain.

However, the invention should not be interpreted as being limited solely to this variant Factor VIII; rather, the invention encompasses other variants where amino acids have been removed to increase release of Factor VIII protein into the systemic circulation. The term "skin cells," as used herein, includes suprabasal and basal keratinocytes.

In a preferred embodiment, the nucleic acid encoding Factor VIII construct of the invention comprises several DNA elements. These DNA elements include a sequence encoding human Factor VIII (preferably the "VIIILA" 4.7 kb cDNA where amino acids 741 through 1639 have been deleted and which DNA sequence encodes a precursor protein of approximately 165 kDa), comprises a promoter

/regulatory sequence, an intron sequence, any necessary 5' or 3' untranslated regions which flank DNA encoding Factor VIII, or a biologically active fragment thereof, and a polyadenylation signal adjacent to the 5' end of the human Factor VIII sequence. In another preferred embodiment, the nucleic acid encoding Factor VIII construct comprises an additional mutation wherein -DNA encoding amino acids 741 through 1666 of the Factor VIII protein has been deleted, thereby causing Factor VIII to be expressed as a single chain. This form of Factor VIII is termed "VIELAΔ". The isolated nucleic acid of the invention comprises 5' and 3' untranslated regions of DNA which flank the human Factor VIII cDNA sequence. In one Factor VIII construct exemplified in the experimental examples section, i.e., pinvVIIILA, the 5' untranslated region flanking the human factor sequences is as follows: At the 5' end of the Factor VIII sequences, the human involucrin promoter including a 1.2 kb intron critical for tissue-specific expression, with, at its 3' end, the

SV40 intron sequence. At the 3' end adjacent to the Factor VIII cDNA, the SV40 polyadenylation signal.

It will be appreciated that other 5' and 3' untranslated regions of DNA may be used in place of those recited in the case of human Factor VIII, particularly when DNA encoding proteins other than human Factor VIII is used in the vector of the invention.

Further, the invention should be construed to include naturally occurring variants or recombinantly derived mutants of wild type human Factor VIII DNA sequences, which variants or mutants render the protein encoded thereby either as therapeutically effective as full-length human Factor VIII, or even more therapeutically effective than full-length human Factor VIII in the gene therapy methods of the invention.

For example, chaperone protein BiP/GRP78K sequesters human Factor VIII. Some of the human Factor VIII so introduced is therefore not available for participation in blood coagulation because it is retained intracellularly. Accordingly, the present invention describes introduction of a mutation into the sequence of human Factor VIII cDNA such that the protein encoded thereby does not bind BiP/GRP78K. More specifically, the experimental examples describe introduction of a mutation at the DNA sequence encoding amino acids 291-309 which reduces binding of human Factor VIII protein to BiP/GRP78K.

Such mutants may be useful in the gene therapy methods of the invention for the treatment of hemophilia in that they encode a form of human Factor VIII which may be more easily secreted into the circulation. Preferably, a mutant human nucleic acid encoding Factor VIII which encodes a human Factor VIII protein comprising the amino acid serine in place of phenylalanine in the three-hundred ninth (309th) amino acid position from the beginning of the precursor protein, is useful in the vector-of the invention to reduce or eliminate binding of human Factor VIII to BiP/GRP78K.

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Example 1 : Generation of Transgenic Mice that Express Human Factor VIII in the Skin

The experiments presented in this example may be summarized as follows. Transgenic mouse lines were generated which directed expression of human

Factor VIII in the mouse skin.

A transgenic mouse model was used in these studies because this strategy permits uniform, maximal and sustained expression of the Factor VIII transgene of interest. The transgenic mouse model will allow for controlled and standardized comparisons to determine the optimal parameters for Factor VIII expression in the epidermis and its subsequent delivery to the circulation. Constraints which may otherwise have obscured such comparisons in a non-transgenic system, e.g., variation in gene transfer efficiency, size of target site, or duration of expression, were thereby avoided. Transgenic mice expressing the human nucleic acid encoding Factor VIII were generated as follows. First, a plasmid vector, pH3700-pL2, previously described by Carroll et al. (1993, Proc. Natl. Acad. Sci. USA 90:10270-10274; 1995, Cell 83:957-968) was obtained. The plasmid vector contains an expression cassette wherein the human involucrin promoter sequence was used to drive expression of the gene of interest in suprabasal keratinocytes. Second, a shortened version of the human Factor VIII cDNA, i.e., Factor VIIILA cDNA, derived from plasmid pMT2-LA (toole et al, 1986, Proc. Natl Acad. Sci. USA 83:5939-5942) was cloned into the unique Notl site of-pH3700-pL2. The LA variant of Factor VIII cDNA encodes a protein which lacks most of the "B" domain since amino acids 741 - 1639 are missing from the protein.

Thus, the transgene construct, termed "pinvVIIILA", contained a shortened version of the human Factor VIII cDNA placed under the transcriptional control of the human involucrin promoter. The construct also included an SV40 intron and an SV40 polyadenylation sequence immediately flanking the Factor VIIILA insert. The expression cassette was excised from the pinvVIIILA vector backbone and the DNA was microinjected into the male pronucleus of fertilized mouse oocytes. Forty-six mice were produced and of these, thirteen were identified as carrying the human Factor VIIILA DNA by Southern blot analysis of DNA obtained from tail tissue. Of the thirteen transgenic founder (F0) mice, ten were identified which expressed Factor VIII RNA by RT-PCR analysis of total tail tissue RNA. The ten transgenic mice were then bred with non-transgenic but otherwise identical mice to propagate the transgene. Seven mice were able to pass the transgene to their off-spring, thus, seven separate transgenic mice lines were thus established all of which expressed the nucleic acid encoding Factor VIII in the skin. The Materials and Methods used in the experiments presented in this example are now described.

Construction of pinvVIIILA transgene vector

Recombinant DNA techniques were performed using standard methods as described by Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York; Ausubel et al, 1997, Current Protocols in Molecular Biology. Green & Wiley, New York.

A transgene construct designed to express human Factor VIII, termed pinvVIIILA and depicted in Figure 3, was assembled using an expression cassette containing the promoter for the human involucrin gene, which is normally expressed in suprabasal keratinocytes.

The pH3700-pL2 cloning vector described by Carroll et al. (1993, Proc. Natl. Acad. Sci. USA 90:10270-10274; 1995, Cell 83:957-968), was used to generate the pinvVIIILA transgene expression construct. The pH3700-pL2 plasmid contained the involucrin promoter (3.7 kb), SV40 splice donor-acceptor sequences, Not I cDNA cloning sites, and SV40 polyadenylation signal sequences arranged in tandem from 5' to 3', respectively. Of note, the construct included a 1.2 kb intron occurring at the 3' end of the involucrin promoter region which is essential for tissue-specific expression. The previously described variant of the human Factor VIII cDNA which lacks most of the "B" domain of the native protein, i.e., VIIILA, was cloned into the

Notl site of pH3700-pL2. By way of further explanation, the structures of both the normal full-length Factor VIII protein and the "B" domain-deleted Factor VIIILA variant protein are depicted in Figures 1 and 2.

Human Factor VIII is synthesized as a 2351 amino acid (265 kDa) precursor protein composed of several domains - "A", "B", and "C" ~ categorized on the basis of their homology to each other (Vehar, et al, 1994, Nature 312:337-342). Three "A" domains interrupted by a unique, "non-functional" "B" domain comprise much of the N-terminus and central portion of Factor VIII. Two "C" domains make up the C-terminus. Further, an acidic domain is situated between the N-terminal "Al" and "A2" domains, i.e., acidic 1, and another is located between the "B" and "A3" domains, i.e., acidic 2 (Figure 1).

As discussed previously, human Factor VIII undergoes extensive intracellular post-translational modification through N-glycosylation of asparagine residues and sulfation of tyrosine residues (Kaufman et al, 1988, J. Biol. Chem. 263:6352-6362; Pittman et al, 1992, Biochem. 31 :3315-3325; 1994, J. Biol. Chem. 269:17329-17337). Within the Golgi apparatus, partial cleavage of the "B" domain generates a heavy chain and a light chain which are secreted (Figure 1), circulate and are further cleaved as explained previously. The particular variant, VIIILA cDNA, used in this experiment was selected because the "B" domain deletion has been well-defined and because of the availability of reliable reagents for studying the expression of the human protein encoded by this variant. As noted previously amino acids 741-1639 have been deleted from this variant (Toole et al, 1986, Proc. Natl. Acad. Sci. USA 83:5939-5942). The absence of most of the "B" domain does not compromise Factor VIII expression or activity (Toole et al, supra; Eaton et al, 1986, Biochem. 25:8343-8347; Bihoreau et al, 1995, Biochem. 277:23-31). Instead, removal of the "B" domain simplifies post- translational modification of the Factor VIII protein as 19 of 25 potential N- glycosylation sites are eliminated by the deletion. Moreover, deletion of the "B" domain shortens the human Factor VIII cDNA to roughly 4.7 kb and reduces the predicted protein product to about 165 kDa instead of the normal 265 kDa protein. Without wishing to be bound by theory, this reduction in protein size may facilitate Factor VIII secretion across the basement membrane zone and into the systemic circulation. Moreover, despite removal of most of the "B" domain, the proteolytic cleavage site at residue 1648 is retained in the variant. Therefore, expression of the

VIIILA cDNA generates a precursor peptide that will undergo cleavage yielding a functional but truncated heavy chain and a normal light chain.

The VIIILA cDNA was cloned into the pH3700-pL2 expression cassette as follows. A 4.7 kb fragment containing the VIIILA cDNA was liberated from the pMT2-LA plasmid (Toole et al, 1986, Proc. Natl. Acad. Sci. USA 83:5939-5942) by Xhol-Sall digestion. The VIIILA cDNA ends were made blunt and Notl linkers were attached to facilitate cloning into the Notl sites of the pH3700-pL2 plasmid. Thus, the resulting construct, pinvVIIILA, places the VIIILA cDNA under the transcriptional control of the involucrin promoter as depicted in Figure 3. Transgene sequences were liberated from the vector backbone by Sail digestion prior to oocyte pronuclear injection. Oocyte injections and reintroduction into surrogate female mice were performed by the Transgenic Mouse Core Facility at the University of Pennsylvania according the standard methods described in, for example, Hogan et al. (1986, Manipulating the Mouse Embryo: A Laboratory Manual.

Cold Spring Harbor, New York).

Detection of VIIILA DNA in Founder Mice

Genomic DNA derived from the tail tissue (which is composed mostly of skin) of forty-six founder ("F0") mice was subjected to Southern blot analysis using a 1.7 kb probe specific for the 5' end of the 4.7 kb Factor VIII LA cDNA (Figure 3).

Thirteen transgenic founder mice having the VIIILA gene were identified.

Southern blot analysis

Genomic DNA derived from tail tissue was digested with BglE and size fractionated on a 1.0 % agarose gel. Denatured DNA was then transferred to positively charged Nylon membrane and hybridized with a probe.

RT-PCR Assay for Expression of VIIILA RNA in Founder Mice

In order to identify which, if any, of the transgenic founders were expressing the VIIILA gene, total RNA obtained from tail tissue of the 13 transgenic founders was subjected to RT-PCR analysis. The 5' primer used for RT-PCR analysis was based on 5' untranslated sequences derived from the involucrin promoter region, while the 3' primer was derived from the Factor VIII coding region as shown in Figure 3. Also, the RT-PCR primer pairs flanked both the 1.2 kb involucrin and SV40 intron sequences (Figure 3). Therefore, this primer pair was expected to give rise to a transgene-specific, message-specific RT-PCR amplification product of 280 bp. The Results of the experiments presented in this example are now described.

Thirteen transgenic mice were generated which contained the DNA for human Factor VIIILA cDNA per Southern blot analysis. In all, ten of the thirteen pinvVIIILA founder mice were shown to express the Factor VIII transgene by RT-PCR analysis of total RNA derived from tail tissue. These mice were then mated with nontransgenic but otherwise identical mice to propagate the transgene, and seven founder mice were able to transmit the Factor VIII transgene to the FI generation. These results are summarized in Table 1.

Table 1

Construct pinvVIIILA

# Transgenic Founders 13/46

# Founders Expressing Transgene 10

# Founders Transmitting Transgene 7

Example 2: Generation of Transgenic Mice that Express Human Factor VIII in the

Epidermis But Not at the Normal Sites of Endogenous Factor VIII Expression

The experiments presented in this example may be summarized as follows.

Transgenic mice expressing human Factor VIII were bred with Factor VIII knockout mice, . e. , mice which contained a null allele for the murine nucleic acid encoding Factor VIII and which did not express murine Factor VIII in any tissue including the liver which is the normal site of Factor VIII production. This experiment was conducted in order to generate mice which expressed human Factor VIII in the epidermis but did not express either human or murine Factor VIII where this protein is normally expressed. Thus, an animal model was developed wherein specific tissue targeting of human Factor VIII expression using the involucrin promoter could be observed in a background which was devoid of endogenous Factor VIII expression.

Two lines of Factor VI knockout mice have been previously described which were developed through targeted disruption of either exon 16 or exon 17 of the murine nucleic acid encoding Factor VIII to generate null alleles (Bi et al, 1995, Nat.

Genet. 10:119-121). Murine Factor VIII protein is not detectable by immunoassay using antibodies directed against murine Factor VIII light chain in either of these knockout mice lines (Bi et al, 1996, Blood 88:3446-3450). Mice from both knockout lines are unable to clot blood in response to mild trauma-induced bleeding and exhibit a spontaneous death rate of roughly 5-10% due to hemorrhage (Turecek et al, 1997, Abstract presented at XVI Congress of the International Society on Thrombosis and Haemostasis, 77 (Abstracts 230, Abstract No. P5-938) note, female mice which were bred to homozygosity for the Factor VIII null allele were capable of carrying litters to term and survived giving birth.

Factor VIII Transeenic/Knockout Mouse Matings

The strategy employed for breeding the pinvVIIILA transgene into a knockout background was to mate male transgenic mice with female mice homozygous for the Factor VIII exon 16 null allele (Figure 4). As shown in Figure 4, all male mice progeny derived from this mating would be predicted to be deficient for the endogenous murine nucleic acid encoding Factor VIII since they inherit the X chromosome from their homozygous null-allele mother. Half of these male progeny mice would be predicted to inherit the Factor VIIILA transgene from their father, assuming integration of transgene sequences into an autosomal chromosome. Genotype determination to identify male mice that had the transgene was performed by PCR analysis of tail-derived genomic DNA samples using the aforementioned PCR primers. Transgenic mice from each line were bred to female mice homozygous for the murine Factor VIII null gene (Figure 4).

Localization of Factor VIII protein to the Epidermal Compartments by Immunostaining

Commercially available (American Diagnostica, Inc., Greenwich, CT) ESH2 polyclonal antibodies are used for immunostaining studies. Neonatal mice are sacrificed in order to harvest skin explants to generate blocks for tissue sectioning.

Neonatal mouse epidermis is used because it is several cell layers thick whereas adult mouse epidermis is too thin to distinguish epidermal compartments readily on microscopy. Genotype analysis is performed on tail-derived genomic DNA by PCR to identify transgenic samples as previously described herein. Skin samples from nontransgenic litter mates serve as negative controls.

The Materials and Methods used in the experiments presented in this example are now described. Factor VIII Transgene Expression Analysis

Transgenic mice having the human Factor VIII DNA in a null allele background were identified by Southern blot analysis of tail tissue DNA as previously set forth herein. The tissue specificity of transgene expression was determined by RT- PCR assay of total RNA derived from various tissues. Figure 5 depicts the results of RT-PCR analysis performed on total RNA derived from multiple tissues from a transgenic mouse from line 10. Control RT-PCR reactions for RNA integrity using primers specific for mouse glyceraldehyde phosphate dehydrogenase (GAPDH) message were performed but are not shown herein. Phenotype Correction Analysis Phenotype correction of Factor VIII deficiency was assessed by snipping a segment of tail and determining whether mice were able to clot blood and survive. More specifically, a 0.5 cm segment of tail tissue was snipped to create a uniform wound for each mouse after inducing anesthesia with the inhalant metofane. Measurement of Factor VIII Activity in Blood Plasma The COAMATIC® assay (Chromogenix, Inc., Molndal, Sweden, distributed by Diaopharma, Inc., Franklin, OH) which measures Factor VIE-dependent activation of factor X in a photometric fashion in vitro, was used to assess circulating functional Factor VIII in transgenic mice. Plasma samples obtained from 2 or 3 transgenic mice from each pinvVIIILA line were tested. Samples of plasma were incubated at 37°C with a mixture of factor IXa, factor X, calcium, phospholipids, and a chromogenic substrate for activated factor X. As the reaction progressed, factor X became activated and acted upon its substrate to release a chromophore which absorbs at 405 nm. Thus, the A₄₀₅ measured after termination of the reaction directly correlated with Factor VIII activity in the plasma sample. A standard curve was generated from pooled plasma obtained from normal mice and plasma obtained from a non-transgenic knockout mouse was used as a negative control.

The Results of the experiments presented in this example are now described. Factor VIII Transgene is Expressed in Squamous Epithelium But Not in

Other Tissues

Tissue-specific transgene expression in the skin of Factor VIE/knockout mice was demonstrated. There was also evidence of transgene expression in the tongue and esophagus, but expression was not observed in other tissues including the brain, thymus, lung, heart, liver, intestine, colon, kidney, bladder, and testes. Therefore, expression of the transgene was limited to stratified squamous epithelia and the presence of some degree of transgene expression particularly in the upper GI tract is consistent with observations in other transgenic mouse models generated using the involucrin promoter (Carroll et al, 1993, supra; 1995, supra). Therefore, expression of the transgene in the Factor VIE/knockout transgenic mice in tissues other than the skin, i.e., in the stratified squamous epithelia of the upper GI, may contribute to the Factor VIII replacement observed in these mice. However, the skin is the preferred source of systemic Factor VIII based on the data presented herein. This concern is addressed by the tissue-grafting experiments described below. Transgenic Mice Exhibit Phenotypic Correction

A total of 17 male mice derived from 5 independent pinvVIIILA lines crossed with homozygous null allele knockout female mice were tested in this study (Table 2). All 7 of 7 transgenic (T^") mice, but none of their 10 non-transgenic (T^") but otherwise identical litter mates, were able to clot. These results suggest that Factor VIII expression targeted to the suprabasal epidermis corrected the hemophilia A phenotype. Table 2

Transgenic line #6 #9 #10 #29 #31 Total

T T- r T- T T- T T- T T r T-

# Mice Assayed 2 2 1 1 1 3 1 2 2 2 7 10

# Mice Corrected 2 0 1 0 1 0 1 0 2 0 7 0

Transgenic Mice Exhibit Detectable Factor VIII Activity in Blood Plasma

The average Factor VIII activity in mice from each line was compared with Factor VIII activity of otherwise identical non-transgenic, non-knockout normal mice and the results are shown in the bottom row of Table 3. Factor VIII activity in sera obtained from multiple mice from each line ranged from 6% (line #9) to 32% (line #31) of that found in normal mouse plasma although individual mice from lines #29 and #31 exhibited up to 44% activity of normal mice.

Table 3 Transgenic Line #6 #9 #10 #29 #31

% Factor VIE Activity Compared with Normal Mice

Range 13-19% 6% 8-25% 13-44% 19-44%

Average 15% 6% 19% 26% 32%

Therefore, transgenic mice from 5 independent pinvVIIILA lines (lines

6, 9, 10, 29 and 31) exhibited both phenotype correction and detectable Factor VEI activity in plasma samples. These results demonstrate that expression of nucleic acid encoding Factor VIII targeted to the suprabasal compartment of the epidermis is useful for treatment of hemophilia A through synthesis and secretion of functional Factor VIII that can access the circulation.

Example 3: Generation of Transgenic Mice that Express Factor VIII as a Single Peptide The experiments presented in this example may be summarized as follows.

As discussed previously herein, Factor VIII is normally expressed as two chains derived from a common precursor peptide. Both chains must access the dermal vasculature from the epidermis in order to reconstitute functional Factor VIII in the systemic circulation upon activation by thrombin. Thus, two unstable chains must migrate through the basement membrane zone and into the dermis in order to access the vasculature of the skin and reconstitute Factor VIII activity in the circulation. Expression as two chains, therefore, may be an inefficient way of delivering Factor VIII from the epidermis to the circulation. Thus, without wishing to be bound by theory, epidermal expression of Factor VIII as a single, larger polypeptide which combines the heavy and light chains yet retains the appropriate proteolytic cleavage site necessary for further processing may improve Factor VIII access to the vasculature. Bihoreau et al. (1995, Biochem. 277:23-31), have shown that a variant of Factor VIII in which amino acids 771-1666 have been deleted is not cleaved into a heavy and a light chain. Further, Bihoreau et al, supra, demonstrated that the variant, termed VIIIΔII, retains activity and several other important properties of VIII in an in vitro system.

The novel variant of Factor VIII described herein extends the deletion of the "B" domain an additional 81 bp (27 amino acids) as shown in Figure 6. Sequences encoding amino acids 741-1666 are absent from this DNA, termed "VIELAΔ," thereby removing both proteolytic cleavage sites at amino acid residues 1313 and 1648. Therefore, this variant of Factor VIII should be expressed as a single, "B" domain- deleted protein which will not be cleaved within the Golgi apparatus but which retains all of the thrombin (factor Ea) cleavage sites required for activation in the circulation.

Instead, this Factor VIII variant should remain a single polypeptide until it reaches the circulation and is activated by thrombin (factor Ea) cleavage at amino acid 740 (Figure 1). This novel version of Factor VIII is similar to but even smaller than the VIIIΔE cDNA, which lacks amino acids 771-1666, gives rise to a single uncleaved Factor VIII peptide, and retains functional activity in vitro (Bihoreau, et al, 1995, supra).

To assess the delivery of functional Factor VIII across the basement zone and into the dermal vasculature as a single polypeptide, the VEILAΔ cDNA was cloned into the pinvVIIILA construct previously described herein. This construct was designated as "pinvVIELAΔ."

The Materials and Methods used in the experiments presented in this example are now described.

Construction of Factor VIELAΔ Transgene Factor VIIILA cDNA was used to extend the deletion in the "B" domain by an additional 81 bp. The strategy used to further delete portions of the "B" domain is illustrated in Figure 6. Briefly, the VIELAΔ cDNA clone was generated by introducing a deletion of the 81 bp encoding amino acids 1640 to 1666 into the VIIILA cDNA using a PCR-based strategy. One set of PCR primers was designed to amplify the region between the 5' border of the deletion and a unique Kpn I site 0.5 kb further upstream. A second set of primers was designed to amplify sequences between the 3' boundary of the deletion and a unique Apa I site 1.2 kb further downstream. High fidelity Pfu polymerase was used in the PCR reactions to generate amplification products with blunt ends. The upstream and downstream fragments were digested with Kpnl and Apal, respectively, to generate the appropriate sticky end on either side while maintaining the blunt end bordering the 81 bp deletion. Both fragments were cloned into Kpn I- Apa I-digested PMT2-LA plasmid by virtue of a three-way ligation that linked the ends of the 81 bp deletion through a blunt terminal ligation. Appropriate incorporation of the deletion and the integrity of PCR-amplified segments were confirmed by DNA sequence analysis performed according to standard procedures.

The VIELAΔ cDNA was cloned into the Not I sites of the pinvVIIILA plasmid, replacing the VIIILA cDNA. The resulting construct, termed "pinvVEILAΔ," places the VEILAΔ cDNA under the transcriptional control of the involucrin promoter. Generation of pinvVIELAΔ Transgenic Mice In order to generate transgenic mice, pinvNIELAΔ transgene sequences were liberated from the vector backbone by Sail digestion and used for oocyte pronuclear injection as described previously herein.

The Results of the experiments presented in this example are now described.

Generation of pinvNIELAΔ Transgenic Mice

A total of 45 founder mice were obtained following microinjection of the pinvNIELAΔ construct. Genotype determination was performed by Southern blot and PCR analysis of tail-derived genomic DΝA samples from each mouse as described previously. In all, 8 pinvVIELAΔ transgenic founder mice were identified.

Example 4: Generation of Modified Factor VIII Protein Which Does Not Bind the Factor VIII Chaperone Protein BJP/GRP78

The experiments presented in this example may be summarized as follows. It has been recently demonstrated that mutations in full-length Factor

VIII which lead to diminished interaction with the chaperone protein BiP/GRP78 (immunoglobulin Binding Protein/glucose-regulated protein of 78 kDa) improve secretion of Factor VIII in a cell-culture system (Swaroop et al, 1997, J. Biol. Chem. 272: 24121-24124). Altering BiP/GRP78 interaction with Factor VIII, therefore, may improve the efficiency of Factor VIII secretion from the epidermis in vivo.

Secretion of Factor VIII from the cell is inefficient due to its association with BiP/GRP78 (Dorner et al, 1989, J. Biol. Chem. 264: 20602-20607). BiP/GRP78 binds to polypeptide folding intermediates of selected secreted proteins, such as Factor VIII, in the lumen of the endoplasmic reticulum. Release of peptides from BiP/GRP78 to permit transit to the Golgi apparatus requires high levels of intracellular ATP and

BiP/GRP78 ATPase -activity (Munro and Pelham, 1986, Cell 46: 291-300; Dorner et al, 1990, Proc. Natl. Acad. Sci. USA 87: 7429-7432; Dorner and Kaufman, 1994, Biologicals 22: 103-112). Stable association with BiP/GRP78 may occur for misfolded or incompletely N-glycosylated proteins, thus inhibiting their secretion (Dorner et al,

1987, J. Cell Biol. 105: 2665-2674; and 1988, Mol. Cell Biol. 8: 4063-4070). Expression of BiP/GRP78 mRNA and protein are induced by the presence of misfolded or incompletely glycosylated polypeptides in the endoplasmic reticulum (Kosutsurni et al, 1988, Nature 322: 462-464; Dorner, et al, 1989, supra; and Lee, 1992, Curr. Op. Cell Biol. 4: 267-273). Further, overexpression of selected secreted proteins, including Factor VIII, in a cell culture model induces BiP/GRP78 expression in vitro (Dorner et al, 198-9, supra). Additionally, the efficiency of secretion of Factor VIII and other proteins is inversely correlated with levels of BiP/GRP78 expression (Dorner, et al,

1988, supra; and 1992, EMBO J. 11 : 1563-1571).

Therefore, it has been proposed that BiP/GRP78 functions to sequester abnormal proteins to prevent their transit from the endoplasmic reticulum, particularly under conditions of physiological stress which may predispose the cell to generate abnormally configured-proteins.

Approximately 80-85% of full-length Factor VIII within a cell is tightly bound to BiP/GRP78 in the endoplasmic reticulum; thus, only a fraction of the Factor VIII protein synthesized is ultimately secreted (Dorner et al, 1987, supra). Interfering with BiP/GRP78 binding Factor VIII may, therefore, improve the efficiency of Factor VIII secretion. Indeed, only 60% of "B" domain-deleted Factor VIII remains bound to

BiP/GRP78 (Dorner et al, 1987, supra). As noted previously, 19 of 25 potential N~ glycosylation sites have been deleted in this Factor VIII variant. Presumably, this reduced requirement for N-glycosylation limits BiP/GRP78 sequestration of Factor VIII protein. BiP/GRP78 has been predicted to bind a heptameric unit, which mediates its interaction with selected proteins (Blond-Elguindi et al, 1993, Cell 75: 717-728).

BiP/GRP78 binding to Factor VIII occurs within a 110 amino acid region localized to the Factor VIII Al domain (Marquette, et al, 1995, J. Biol. Chem. 270: 10297-10303). Within this segment of the Al domain, residues spanning He²⁹¹ to Phe³⁰⁹ comprise a hydrophobic cluster which contains heptamers which show a high probability of binding to BiP/GRP78 (Marquette, et al, 1995, supra). Mutation of Phe³⁰⁹ to serine or alanine in full-length Factor VIII reduces Factor VIII binding to BiP/GRP78, improves its secretion, and retains its functional activity in vitro (Swaroop, et al, 1997, J. Biol. Chem. 272: 24121-24124). Because of the potential for increased efficiency of Factor VIII secretion due to decreased binding to BIP/GRP78, a variant of the "B" domain-deleted human Factor VIIILA cDNA is produced which contains a Phe³⁰⁹ to Ser³⁰⁹ mutation known to decrease Factor VIII binding to BIP/GRP78. The transgene construct is used to generate transgenic mice that direct its expression to the suprabasal epidermal compartment as described previously. Further, the transgene is propagated into a

Factor VIII knockout background and transgene expression, circulating Factor VIII protein levels, Factor VIII activity, and phenotype correction are determined as described previously. The Materials and Methods used in the experiments presented in this example are now described. Generation of Factor VIIILA Variant Having a Mutation from Phe³⁰⁹ to

Ser³⁰⁹

A variant of the "B" domain-deleted VIIILA cDNA is generated which incorporates a Phe³⁰⁹ to Ser³⁰⁹ mutation (Figure 8). Primers for PCR amplification of the region spanning Phe³⁰⁹ and a unique Spel site upstream are generated. The reverse primer initiates at codon 315 and contains a single base change that converts codon 309 from "TTT" (phe) to "TCT" (ser). A forward primer starting at codon 316 and a reverse primer containing a unique downstream Kpnl site have been synthesized as well. Both sets of primers are used for PCR amplification of their respective regions using Pfu polymerase to achieve high fidelity amplification and to generate blunt ends. The upstream 426 bp fragment contains a 5' Spe I site and a 3' blunt end. The downstream 823 bp fragment has a 5' blunt end and a 3' Kpn I site. After appropriate restriction digestion, both fragments are included in a three-way ligation with Spe I- Kpn I-digested VIIILA cDNA (Toole, et al. 1986, supra). The PCR-amplified fragments are ligated to the plasmid at their sticky ends and to each other at their blunt ends. The resulting product (NIELAP309S) reconstitutes in-frame Factor VIIILA coding sequence and contains the Phe³⁰⁹ to Ser³⁰⁹ mutation. All PCR-amplified regions and cloning junctions are sequenced to confirm their integrity.

The Results of the experiments presented in this example are now described.

To determine the in vivo effect of the Phe³⁰⁹ to Ser³⁰⁹ mutation on secretion of "B" domain-deleted Factor VIIILA from the epidermis, transgenic mice are generated which target expression of the VIELAP309S cDΝA to the epidermis as described previously for pinvVIIILA. That is, the VIIILAP309S cDΝA is placed under the transcriptional regulation of the involucrin promoter by inserting it into the pinvVIIILA construct described previously herein, in place of the VIIILA cDΝA. Thus, transgenic lines which direct VIELAP309S expression to the suprabasal compartment are generated.

The VIELAP309S transgenic lines are bred into the Factor VIII knockout background described previously herein, and transgene expression is characterized by evaluating circulating Factor VIII protein levels and activity, and assessing phenotype correction all as described previously herein. These parameters are compared among VEILAP309S transgenic mice and transgenic mice expressing other Factor VIII variants to determine whether enhanced levels of Factor VIII secretion can be achieved by diminishing Factor VIII binding to BiP/GRP78.

Example 5: Phenotype Correction by Focal Skin Source of Factor VIII

The experiments presented in this example may be summarized as follows.

In order to demonstrate that epidermal-specific delivery of functional human Factor VIII to the systemic circulation can be accomplished in the absence of any ectopic gene expression, tissue grafting experiments are performed. As discussed previously herein, Factor VIII transgene expression driven by an involucrin promoter was detected in both skin and the in tissues of the upper GI tract lined by stratified squamous epithelium. In the aforementioned experiments, the vast majority of Factor VIII expression observed was probably derived from the -epidermal expression of the transgene due to the large surface area of the skin compared to the surface area of the internal stratified squamous epithelia. Further, other stratified squamous epithelial sites are subject to similar barriers to transgene product access to the subepithelial vasculature as is found in the epidermis. Thus, it is unlikely that other epithelial sites deliver Factor VIII to the circulation more efficiently than the epidermis. Nonetheless, Factor VIII expression at other sites may contribute to phenotype correction in the experiments discussed previously. Therefore, the experiments described below are designed to effectuate the delivery of Factor VIII exclusively from a focal cutaneous source through transfer of skin explants from transgenic mice to Factor VIE-deficient null-allele immunocompromised mice. These experiments are an important model for demonstrating the feasibility of Factor VIII transgene therapy via a skin delivery system and for optimizing the system to, eventually, treat humans afflicted with hemophilia A.

Generation of Factor VIE/RAGl Double Knockout Mice The Factor VIII exon 17 knockout allele was bred into a RAG-1 knockout, immunodeficient background to generate recipient mice that would not reject transplanted grafts. The recombination activation gene-1 (RAG-1) product functions in VDJ recombination at both immunoglobulin and T cell receptor loci. Thus, RAG-1 knockout mice generate no mature B or T lymphocytes and are therefore immunodeficient (Mombaerts et al, 1992, Cell 68:869-877). Immunocompromised male mice homozygous for the recombination activating gene-1 (RAG1) knockout allele (Jackson Labs) were mated with female mice homozygous for a Factor VIII null allele. Thus, the "double knockout" progeny mice are both Factor VIII deficient (Bi et al, 1995, Nat. Genet. 10:119-121) and immunodeficient (Mombaerts et al, 1992, Cell 68:869-877). More specifically, all FI female mice would be heterozygous for the null alleles at both loci, and all FI male mice would be heterozygous for the RAG1 null allele, yet they would be Factor VIE-deficient given inheritance of the maternal X - chromosome carrying the Factor VIII null allele. FI male and FI female mice were mated to yield F2 mice as double knockout mice.

Grafting of Factor VIE-expressing Skin from Transgenic Mice onto Factor VIE/RAGl Double Knockout Recipient Mice

Adult Factor VIE/RAGl double knockout mice are anesthetized. Plasma-derived human Factor VIII protein (Armour Pharmaceutical Co., Kankakee, IL) at a dose of 2.5 units (roughly 2.5 times normal levels for mice) is prophylactically administered through intravenous tail vein injection to minimize bleeding during the grafting procedure. Human Factor VIII protein has been reported to have a half life of less than 1 hour in mice (Hoeben et al, 1993, Hum. Gen. Ther. 4: 179-186). It was established herein that all Factor VIE/RAGl double knockout mice that received this dose of human Factor VIII survived surgery and demonstrated no detectable Factor

VIII activity after 1 week.

After the back hair is shaved and the skin is treated with a topical delipatory agent, the back skin is sterilized with 70% ethanol and betadine. A grafting site is prepared by dissecting skin to the level of panniculus carnosa. A full-thickness explant derived from a "high-expressing" transgenic donor is placed, dermal-side- down, on the wound and secured with 6.0 ethilon sutures. Triple antibiotic ointment is then placed on the graft. Tegaderm adhesive dressing, zinc oxide-impregnated gauze and elastic Coban gauze are applied in sequence and the outer Coban layer is secured using surgical staples. Grafts remain dressed for 3 weeks. Plasma samples are collected by tail bleeding prior to grafting and each week after grafting. Circulating Factor VIII levels and activity are measured as described previously. Phenotype correction is determined at 4 to 8 weeks after grafting using the whole blood clotting assay. Mice that receive grafts from non- transgenic, Factor VIE-deficient knockout mice are used as negative controls for Factor VIII activity assays and phenotype correction studies.

The Results of the experiments presented in this example are now described. Factor VIE/RAGl Double Knockout Mice

F2 mice have been generated and genotyped using PCR primers specific for the null and wild type alleles for both RAG1 and Factor VIE null allele loci (Figure 7). It was statistically predicted that one of eight male mice and one of eight female mice would be a double knockout. To date, several male and female double knockout mice have been identified and many breeding pairs are mating to propagate this line.

Characterization of Recipient Factor VII Expressing Skin Grafts

In order to further investigate the parameters required for successful phenotype correction through grafting explants, skin explants derived from high Factor VEI-expressing mouse lines that direct Factor VIII production to the suprabasal epidermal compartments, are grafted onto adult double knockout Factor VIII defϊcient/RAGl mice.

The Results of the experiments presented in this example are now described.

Off spring of matings between male and female transgenic mice from high-expressing lines were used as skin donors. Skin explants were placed on recipient sites dissected to the panniculus carnosa of Factor VIE/RAG- 1 double knockout mice. Mice that received grafts were given an intravenous injection of human Factor VIII peri-operatively to prevent exsanguination. Human Factor VIII has a half life of less than one hour in mouse blood and was not detectable one week after grafting. Mice that received grafts from non-transgenic Factor VIII knockout mice served as negative controls. Plasma samples were obtained from mice prior to grafting and every one to two weeks thereafter for up to eight weeks.

Two experimental mice have provided useful information. These mice maintained roughly four square cm of transplanted skin. Samples of engrafted skin were obtained to confirm graft viability and transgene expression by RT-PCR analysis for Factor VIII mRNA. Circulating Factor VIII activity of at least about 6% to about 10% was detectable in plasma samples obtained from these mice using the COAMATIC® assay. Phenotypic correction was confirmed by demonstrating that whole blood obtained from these mice clotted in vitro.

These results demonstrate correction exclusively from a cutaneous source of Factor VIII in the absence of expression of other stratified squamous epithelium and correction from a focal source of Factor VIII, thus supporting the feasibility of epidermal targeted Factor VIII gene therapy. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations

Claims

What is claimed is:

1. A composition comprising an isolated human Factor VIE nucleic acid, or a biologically active fragment thereof, wherein said Factor VIII nucleic acid has a first mutation comprising a deletion of a portion of the "B" domain of said Factor VIII nucleic acid, said deletion effecting the removal of both cleavage sites of the heavy and light chains and a part of the acidic 2 domain of the human Factor VIII protein.

2. A composition comprising an isolated human Factor VIII nucleic acid, or a biologically active fragment thereof, wherein a portion of the "B" domain of said human Factor VIII is deleted, said human Factor VIII nucleic acid further comprising a second mutation, wherein said second mutation reduces the ability of the human Factor VIII protein to bind a chaperone protein.

3. The composition of claim 2, where said chaperone protein is immunoglobulin Binding Protein glucose-regulated protein of 78 kDa ("BiP/GRP78").

4. The composition of claim 3, wherein said second mutation effects a non-conservative amino acid substitution at amino acids 291 through 309 of the human Factor VIII protein.

5. The composition of claim 4, wherein said second mutation substitutes a phenylalanine amino acid residue with a serine amino acid residue at. position 309 in said mature human Factor VIII protein.

6. The composition of claim 1, wherein said nucleic acid further comprises a promoter/regulatory sequence operably linked to said human Factor VIII nucleic acid, wherein said promoter/regulatory sequence is positioned at the 5' end of said human Factor VIII nucleic acid.

7. The composition of claim 6, wherein said promoter/regulatory sequence is selected from the group consisting of an inducible promoter, a constitutive promoter and a tissue specific promoter, a basal skin layer specific promoter, a suprabasal skin layer promoter, a keratin promoter, an involucrin promoter, a cytokine promoter, a growth factor promoter, the SV40 early promoter, a retroviral LTR promoter and the cytomegalovirus immediate early promoter.

8. The composition of claim 7, wherein said nucleic acid further comprises a second regulatory sequence positioned between the 3' end of said promoter/regulatory sequence and the 5' end of said human Factor VIII nucleic acid.

9. The composition of claim 8, wherein said second regulatory sequence is an intron.

10. The composition of claim 9, wherein said intron is selected from the group consisting of an SV40 intron, and a rabbit β-globin intron.

11. The composition of claim 9, wherein said nucleic acid comprises a polyadenylation signal sequence positioned at the 3' end of said human Factor VIII nucleic acid.

12. The composition of claim 11, wherein said polyadenylation signal sequence is selected from the group consisting of an S V40 polyadenylation signal sequence, a rabbit β-globin polyadenylation signal sequence and a keratin polyadenylation signal sequence.

13. The composition of claim 11 , wherein said promoter/regulatory sequence is the involucrin promoter, and wherein said second regulatory sequence is the SV40 intron, and further wherein said polyadenylation signal sequence is the SV40 polyadenylation signal sequence.

14. The composition of claim 11, wherein said promoter/regulatory sequence is a keratin promoter, and wherein said second regulatory sequence is the rabbit β-globin intron, and further wherein said polyadenylation signal sequence is a keratin polyadenylation signal sequence.

15. The composition of claim 11 , wherein said promoter/regulatory sequence is a keratin promoter, and wherein said second regulatory sequence is the SV40 intron, and further wherein said polyadenylation signal sequence is the SV40 polyadenylation signal sequence.

16. A composition comprising an isolated human Factor VIII protein, or a biologically active fragment thereof, wherein said protein comprises a first mutation comprising deletion of amino acids 741 through 1666 of the full-length human Factor VIII protein.

17. A composition comprising an isolated human Factor VIII protein, or a biologically active fragment thereof, wherein a portion of the "B" domain of said human Factor VIII is deleted, said human Factor VIII protein further comprising a second mutation which reduces the ability of said human Factor VIII to bind a chaperone protein.

18. The composition of claim 17, wherein said chaperone protein is

BiP/GRP78.

19. The composition of claim 18, wherein said second mutation effects a non-conservative amino acid substitution at amino acids 291 through 309 in the human Factor VIII protein.

20. The composition of claim 19, wherein said second mutation causes the substitution of a phenylalanine amino acid residue with a serine amino acid residue at position 309 in said human Factor VIII protein.

21. A cell comprising an isolated human Factor VIII nucleic acid, or a biologically active fragment thereof, wherein said Factor VIII nucleic acid has a first mutation comprising a deletion of a portion of the "B" domain of said Factor VIII nucleic acid, said deletion effecting the removal of both cleavage sites of the heavy and light chains and a part of the acidic 2 domain of the human Factor VIII protein.

22. The cell of claim 21, wherein said cell is a mammalian cell.

23. The cell of claim 22, wherein said cell is selected from the group consisting of a mouse, a rat and a human cell.

24. The cell of claim 22, wherein said cell is an epidermal cell

25. The cell of claim 24, wherein said cell is selected from the group consisting of suprabasal keratinocytes and basal keratinocytes.

26. The cell of claim 25, wherein said cell is a human cell.

27. A vector comprising an isolated Factor VIII nucleic acid, or a biologically active fragment thereof, wherein said Factor VIII nucleic acid has a first mutation comprising a deletion of a portion of the "B" domain of said Factor VIII nucleic acid, said deletion effecting the removal of both cleavage sites of the heavy and light chains and a part of the acidic 2 domain of the human Factor VIII protein, or a modification or portion thereof.

28. A nonhuman transgenic mammal encoding the human Factor VIII nucleic acid of claim 1, said nucleic acid further comprising a promoter/regulatory sequence operably linked to said human Factor VIII nucleic acid, and further wherein said human Factor VIII nucleic acid is expressed in said transgenic animal, and wherein said transgenic mammal exhibits a phenotypic correction compared with an otherwise identical nontransgenic littermate.

29. The transgenic mammal of claim 28, wherein said mammal is a mouse.

30. The transgenic mouse of claim 29, wherein said phenotypic correction is the shortening of coagulation time of the blood of said mammal compared with the coagulation time of the blood of an otherwise identical nontransgenic littermate.

31. A method of expressing the isolated human Factor VIII nucleic acid of claim 1 in the skin of a mammal wherein said Factor VI is secreted into the circulation of said mammal, said method comprising introducing said isolated nucleic acid into skin cells, culturing said skin cells in vitro, and grafting said skin cells onto said mammal.

32. A method of delivering Factor VIII to the blood of a mammal, said method comprising administering to the skin of said mammal nucleic acid encoding Factor VIII.

33. A method of treating hemophilia A in a mammal, said method comprising obtaining skin cells from a mammal, introducing the isolated human Factor VIII nucleic acid of claim 1 into said cells, culturing said skin cells in vitro, and grafting said skin cells expressing Factor VIII onto said mammal.

34. A method of treating hemophilia A in a mammal, said method comprising obtaining skin cells from a mammal, introducing an isolated human Factor VIII wild type nucleic acid into said cells, culturing said skin cells in vitro, and grafting said skin cells expressing Factor VIII onto said mammal.