WO2000077190A2 - Induction of vascular endothelial growth factor (vegf) by the serine/threonine protein kinase akt - Google Patents

Abstract

The present invention relates to methods and compositions for therapeutic angiogenesis. More especially, this invention is directed to induction of vascular endothelial growth factor (VEGF) expression by the serine/threonine protein kinase AKT. Preferably, the compositions and methods according to the invention comprise a nucleic acid encoding AKT, and administration thereof.

Description

INDUCTION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) BY THE SERINE/THREONINE PROTEIN KINASE AKT

FIELD OF THE INVENTION

The present invention relates to methods and compositions for therapeutic angiogenesis. More especially, this invention is directed to induction of expression of the angiogenic protein vascular endothehal growth factor (VEGF) by the serme/threonme protein kmase Akt. Preferably, the compositions and methods according to the invention comprise a nucleic acid encoding Akt, and administration thereof

BACKGROUND OF THE INVENTION Angiogenesis

Angiogenesis is a biological process resulting in the development of new blood vessels The process involves vaπous cell-cell, cell-matπx and cell-cytokme interactions (Mehllo et al., 1997, Cardiovascular Research 35:480-489; Lewis et al., 1997, Cardiovascular Research 35:490-497). Formation of a new vascular network is normally rare in adult organisms. However, new vasculature may occur under a variety of pathologic conditions, including ischemia, inflammation, wound healing, tumor growth, diabetic retmopathy, rheumatoid arthritis, psoriasis and chronic wounds

Therapeutic angiogenesis involves the deliberate stimulation of new blood vessel development using appropπate angiogenic growth factors. Therefore, therapeutic angiogenesis may be used to treat a variety of ischemic conditions or to stimulate wound healing. Ischemic conditions may affect the heart, lower limbs, skm flaps, peripheral nerves, bone, or grafts. Ischemic conditions include cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, peripheral arterial disease, intermittent claudication, ischemic cardiomyopathy and myocardial ischemia (WO97/14307).

A vaπety of factors have been demonstrated to have angiogenic activity. Among these factors are basic and acidic fibroblast growth factors (bFGF and aFGF), FGF-5 (US Pat. 5,661,133), endothehal cell growth factor (Pu et al., 1993, Circulation 88:208-2156), angiopoietm and VEGF (for reviews see Mehllo et al., 1997 and Lewis et al., 1997).

Angiogenesis has also been suggested as essential for the growth and persistence of solid tumors and their metastases (US Pat. 5,854,205). In order to stimulate angiogenesis, tumors may upregulate the production of a vaπety of angiogenic factors, including VEGF. VEGF

Vascular endothehal cell growth factors are a group of angiogenic polypeptides that are members of the platelet-deπved growth factor family of proteins. These protein are glycosylated cationic dimers having a molecular weight of approximately 46-48 kDa. Unlike other angiogenic factors, VEGF is preceded by a natural signal sequence enabling its secretion from intact cells Alternative names for VEGF include vascular permeability factor (VPF) and c-fos induced growth factor (FIGF).

Several forms of VEGF have been identified, including VEGF_]2ι (US Pat. 5,219,739), VEGF₁₆₅ (US Pat. 5,332,672), VEGF₁₈₉ (US Pat. 5,240,848), VEGF₂₀₆, VEGF-2 (W095/24473, W096/39515), VEGF-B (US Pats. 5,607,918 and 5,840,693), and VEGF-D (W097/12972). Various forms of VEGF have been shown to be mitogemc for vascular endothehal cells and to enhance collateral blood vessel formation and blood flow m ischemic tissue.

Transition metal ions, such as CoCl₂, have been shown to enhance expression of the VEGF gene and to stimulate vasculaπzation (US. Pat. 5,480,975). Akt

The AKT proteins are serme/threonme protein kinases that play a role in apoptosis (programmed cell death). Recently, two intracellular signaling pathways involved in the regulation of cell survival/death have been studied Activation of apoptotic stimulating kmase 1 (ASKl) leads to apoptosis in vaπous cell types (Ichijo et al. 1997), while a pathway involving phosphomositide 3- kinase (PI3K) and Akt leads to cytoprotection. It has been demonstrated that the activity of ASKl is induced by tumor necrosis factor-alpha (TNFa) treatment or Fas hgation (Ichijo et al. 1997, Chang et al. 1998) Overexpression of ASKl dominant negative mutants inhibit apoptosis induced by TNFa or Fas hgation, indicating that ASKl plays important roles duπng TNFa or Fas hgation-induced apoptotic cell death. The molecular mechanism by which ASKl induces apoptosis is not clear. It has been shown that ectopic expression of ASKl leads to activation of various stress-activated signaling pathways, such as the MKK4/JNK and MKK6/p38 pathways, which may mediate ASKl -induced apoptosis (Ichijo et al. 1997).

The PI3K/Akt pathway also appears important for regulating cell survival/cell death (Kuhk et al. Franke et al 1997, Kauffmann-Zeh et al, Hemmings 1997. Dudek et al. 1997). Survival factors, such as platelet derived growth factor (PDGF), nerve growth factor (NGF) and msulm-hke growth factor-1 (IGF-1), promote cell survival under vaπous conditions by inducing the activity of PI3K (Ku k et al. 1997, Hemmmgs 1997). Activated PI3K leads to the production of phosphatidylmositol (3,4,5)-tπphosphate (Ptdlns(3,4,5)-P3), which in turn binds to and induces the activity of a AH/PH- domam containing serme/threonme kmase, Akt (Franke et al 1995, Hemmings 1997b, Downward 1998, Alessi et al. 1996). Specific inhibitors of PI3K or dominant negative Akt mutants abolish survival-promoting activity of these growth factors or cytokmes. In addition, mtroduction of constitutively active PI3K or Akt mutants promotes cell survival under conditions in which cells normally undergo apoptotic cell death (Kulik et al. 1997, Dudek et al. 1997). These observations demonstrate that the PI3K/Akt pathway plays important roles for regulating cell survival or apoptosis.

Two isoforms of human Akt protein kmases, Aktl and Akt2, have been identified in the literature (Staal 1987). A third form of human Akt, designated Akt3, is described in US Provisional application number 60/125,108). Yet another isoform of Akt is described m Nakatam et al., 1999 (Biochem. Biophys. Res Comm. 257, 906-910). A rat Akt sequence has also been identified (Konishi et al. 1995)

Seπne-473 in the C-termmus of human Aktl has been shown to be critical for its regulation (Stokeo et al. 1997, Stephens et al. 1998) Upon growth factor stimulation, PI3K is activated. The product of PI3K, Ptdlns(3.4.5)-P binds Aktl, and causes translocation of Aktl from the cytoplasm to the proximity of the inner cytoplasmic membrane, where it becomes phosphorylated at residues Thr308 and Ser473 (Downward, 1998). Phosphorylation of these residues is critical for the activation of Aktl. A recently identified protein kmase, PDK1, has been shown to be responsible for the phosphorylation of Thr308, while the kιnase(s) which phosphorylates Ser473 has not yet been identified (Stokeo et al. 1997, Stephens et al. 1998). Gene Therapy

Gene therapy involves correcting a deficiency or abnormality (mutation, aberrant expression, and the like) by introduction of genetic information mto a patient, such as mto an affected cell or organ of the patient. This genetic information may be introduced either in vitro into a cell, the modified cell then being reintroduced into the body, or directly in vivo mto an appropπate site. In this regard, different techniques of cell transfection and of gene transfer have been descπbed in the literature (see Roemer and Friedman, Eur. J. Biochem. 208 (1992) 211), including transfection of "naked DNA" and various techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), of DNA and hpids (Feigner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J.Biol.Chem. 255 (1980) 10431) and the like. More recently, the use of viruses as vectors for the transfer of genes has emerged as a promising alternative to physical transfection techniques. In this regard, different viruses have been tested for their capacity to mfect certain cell populations, including retroviruses, herpes viruses, adeno-associated viruses, and adenoviruses.

Gene therapy for angiogenesis, specifically employing a sequence encoding a VEGF has been proposed (Mehllo et al., 1997; Lewis et al., 1997). Intraarteπal or intramuscular administration of a plasmid compπsmg the cDNA for VEGF₁₆₅ increases collateral blood flow m an ischemic rabbit hmdlimb model (Tsurumi et al., 1996, Circulation 94:3281-3290; Takeshita et al., 1996, Biochem. Biophys. Res. Commun. 227:628-635). Plasmids comprising the cDNAs for human VEGF₁₂₁NEGF₁₅-, and VEGF_]89 have also been shown to have angiogenic activity m this model (Takeshita et al., 1996, Lab Invest. 75:487-501; WO97/14307) Similarly, a replication defective adenovirus comprising a VEGF₁₆₅ coding sequence induces neovascularization in mice (Muhlhauser et al., 1995, Circ. Res 77. 1077-1086). In human patients, a plasmid comprising a sequence for VEGF₁₆₅ has been shown to induce angiogenesis in ischemic limbs (Isner et al., 1996, Lancet 348: 370-374) and in the heart (see Time magazine, January 11, 1999, pp. 68-73).

The present invention relates to an alternative method for stimulating angiogenesis, rather than by direct administration of a VEGF coding sequence More specifically, Applicants have unexpectedly discovered that VEGF production is induced by the protein Akt. Therefore, the present invention is directed to stimulating expression of a VEGF in a cell by introducing an Akt protein mto the cell. Preferably, the cells are present in a patient suffering from an ischemic condition, and the result is beneficial collateral blood vessel formation in the patient.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for stimulating expression of a VEGF m a cell. More specifically, Applicants have unexpectedly discovered that Akt proteins are able to stimulate VEGF expression. In its most preferred aspect, the cells are present m a patient suffering from an ischemic condition, and the result is beneficial collateral blood vessel formation in the patient

Therefore, a first subject of the invention relates to a method of inducing expression of VEGF m a cell by administering to the cell an Akt protein. The protein may be any Akt protein. Preferably, the Akt protein is a human Akt protein More preferably, the Akt protein is human Aktl, Akt2 or Akt3.

The VEGF produced upon administration of an Akt protein may be any form of VEGF capable of stimulating angiogenesis. Preferably, the VEGF is VEGF₁₂₁, VEGFι₆₅, VEGF₁₈₉, VEGF₂₀6, VEGF-2, VEGF-B, or VEGF-D.

In one aspect, Akt protein is administered to cells. In a preferred embodiment, a nucleic acid encoding an Akt protein, and operatively associated with an expression control sequence, is administered to cells. The nucleic acid may be part of a plasmid or viral vector. Preferred viral vectors are retrovirus, adenovirus, adeno-associated virus, herpes virus, and vaccinia virus.

The Akt protem, or nucleic acid encoding an Akt protein, may be administered alone or in combmation with a transition metal ion and or a vasodilator The Akt protein, or a nucleic acid encoding an Akt protein may also be administered with a nucleic acid encoding a second angiogenic factor operatively associated with an expression control sequence. Preferred angiogenic factors include a VEGF, acidic fibroblast growth factor, basic fibroblast growth factor, endothehal cell growth factor, or an angiopoietm. In another aspect, more than one form of Akt protein may be administered to the cell.

This invention also relates to a method of inducing expression of VEGF in cells of a patient suffering from an ischemic condition by administering to the patient an Akt protein. The ischemic condition may be cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, myocardial ischemia, or ischemic, idiopathic or hypertrophic cardiomyopathy. The protein may be any Akt protein. Preferably, the Akt protein is Aktl , Akt2 or Akt3

The VEGF produced upon administration of an Akt protein may be any form of VEGF capable of stimulating angiogenesis. Preferably, the VEGF is VEGF_12], VEGF₁₆₅, VEGF_]89, VEGF₂₀₆, VEGF-2, VEGF-B, or VEGF-D.

In one aspect, Akt protein is administered to the patient. In a preferred embodiment, a nucleic acid encoding an Akt protein, and operatively associated with an expression control sequence, is administered to the patient. The nucleic acid may be part of a plasmid or viral vector. Preferred viral vectors are retrovirus, adenovirus, adeno-associated virus, herpes virus, and vaccinia virus. In a most preferred embodiment, a nucleic acid encoding the Akt protein is administered directly mto cardiac tissue by transepicardial surgical administration or by percutaneous delivery using a catheter.

The Akt protein, or nucleic acid encoding an Akt protein, may be administered to the patient alone or in combination with a transition metal ion and/or a vasodilator. The Akt protein, or a nucleic acid encoding an Akt protein may also be administered to a patient with a nucleic acid encoding a second angiogenic factor operatively associated with an expression control sequence. Preferred angiogenic factors include a VEGF, acidic fibroblast growth factor, basic fibroblast growth factor, endothehal cell growth factor, or an angiopoietm. In another aspect, more than one form of Akt protein may be administered to the patient.

The present invention also relates to pharmaceutical compositions comprising a nucleic acid encoding an Akt protein, a transition metal and/or a vasodilator and a pharmaceutically acceptable vehicle. The nucleic acid may be part of a plasmid or viral vector. Preferred viral vectors are retrovirus, adenovirus, adeno-associated virus, herpes virus, and vaccinia virus.

In another aspect, the invention relates to a method of inhibiting angiogenesis in a patient suffeπng from a tumor by inhibiting the level of Akt activity m the patient, thereby inhibiting production of VEGF. The level of Akt may be decreased by introducing an Akt antisense nucleic acid mto cells of the patient under conditions wherein the antisense nucleic acid hybridizes under intracellular conditions to an Akt mRNA The level of Akt can also be decreased by introducing an intracellular binding protein, such as a single cham Fv antibody (scFv), that specifically binds Akt into a patient's cell at a level sufficient to bind to and inactivate Akt In another embodiment, Akt activity can be decreased by administering a nucleic acid encoding a dominant negative form of an Akt. Preferably, the antisense nucleic acid, intracellular binding protein or nucleic acid encoding therefor, or dominant negative are administered directly to tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : Construction of activated Akt3 mutant

Figure 1A: Schematic presentation of activated Akt3- Full length coding sequence of human Akt3 was fused in frame with the Myπstylation signal from human Src gene (Myr) in the N- termmal, and fused in frame with the HA-tag m the C-termmus (HA), (see the Examples) Figure IB: Ectopic expression of activated Akt3 in HEK293 cells. HEK293 cells were transfected with either CMV6-MyrAkt3HA or expression plasmid (CMV6) alone. 24 hours after transfections, cell lysates were prepared and subjected to lmmunoblottmg with a-HA antibodies. Figure 1C: Activated Akt3 possesses Akt activity HEK293 cells were transfected with expression plasmid for activated Akt3 (MyrAkt3HA) or expression vector alone (CMV6). 24 hours after transfections, cell lysates were prepared and subjected to lmmunoprecipitation with anti-HA antibodies. Akt3 kmase activities of lmmunopellets were measured by using substrate peptide deπved from GSK3. Bkgd: background level from non-transfected cells; CMV6: CMV6 transfected cells; Akt3cak: cells transfected with expression plasmid for constitutively activated Akt3 (CMV6-MyrAkt3HA). (see the Examples). Figure 2: Akt increases VEGF- 165 secretion from HeLa cells

HeLa cells were transfected with expression plasmid for activated mouse Aktl (Aktl), activated human Akt3 (Akt3) or CMV6 vector alone. One day after transfection, cells were switched to low mitogen media (DMEM-0.5%FBS). 16 hours later, culture media was collected for VEGF ELISA assay. Lane A: cells (6-well tissue culture dish) transfected with 0.4 μg CMV6 vector DNA; Lane Aktl : cells (6-well tissue culture dish) transfected with 0 4 μg CMV6- mAktlcak expression plasmid; Lane Akt3: cells (in 6-well tissue culture dish) transfected with expression plasmid for activated human Akt3 (Akt3). Figure 3 Akt increases VEGF-165 expression m human coronary smooth muscle cells and human skeletal muscle cells

Figure 3A. Human skeletal muscle cells (HSKMCs) were infected with recombmant adenoviruses for green fluorescence protein (AV-GFP), constirutively active mouse Aktl (AV- mAktlcak) or constirutively active human Akt3 (AV-hAkt3cak) at the concentration of 3xl0⁸VP/ml overnight (VP: viral particles). One day after infection, media were collected and VEGF levels in the media were measured by ELISA assay. Figure 3B: Human coronary artery smooth muscle cells (HCASMCs) were infected AV-GFP, AV- mAktlcak, AV-hAkt3cak at the concentration of 3xl0⁸VP/ml overnight. One day after infections, media were collected and VEGF levels in the media were measured by ELISA assay for human VEGF. Figure 3C: HCASMCs were infected with indicated viruses at concentration of 3xl0⁸VP/ml overnight. As a control, non-mfected cells were switched to hypoxia condition. One day later, total RNA was isolated from these cells and VEGF expression were detected by Northern blot analysis. Figure 4. Akt increase VEGF expression in rat cardiomyocytes

Neonatal cardiomyocytes were infected with recombmant adenoviruses for green fluorescence protein (AV-GFP), constirutively active mouse Aktl (AV-mAktlcak) or constirutively active human Akt3 (AV-hAkt3cak) at a concentration of 3xl0'VP/ml overnight. As a control, non-mfected cells were subjected hypoxia treatment for 24 hours. One day after infection, total RNA was isolated and VEGF expression was detected by Northern blot analysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention advantageously provides methods and compositions for stimulating expression of a VEGF m a cell. Therefore, the invention enables the treatment of ischemic disease in patients by providing the means and methodology for stimulating collateral blood vessel formation in ischemic tissue. More specifically, Akt proteins are shown herein to stimulate the expression of the angiogenic protein VEGF. Therefore, a first subject of the invention relates to stimulation of VEGF expression in cells by introducing an Akt protein to the cells. In a preferred aspect, a nucleic acid encoding the Akt protein is administered to cells.

The invention also relates to the treatment of a patient suffeπng from an ischemic condition by administering to the patient an Akt protein. Preferably, a nucleic acid encoding the Akt protein is administered to the patient, and the result is beneficial collateral blood vessel formation in ischemic tissue in the patient. The vaπous aspects of the invention will be set forth in greater detail in the following sections This organization into various sections is intended to facilitate understanding of the invention, and is m no way intended to be limiting thereof Definitions

The following defined terms are used throughout the present specification, and should be helpful understanding the scope and practice of the present invention.

In a specific embodiment, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably withm 5% of a given value or range.

A "nucleic acid" is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyπbonucleic acid (RNA) and polydeoxyπbonucleic acid (DNA), both of which may be smgle-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.

A "gene" refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.

A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

A "vector" is any means for the transfer of a nucleic acid into a host cell. A vector may be a rephcon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstem-Barr and adenovirus vectors, as set forth m greater detail below. Non-viral vectors include plasmids, liposomes, electrically charged hpids (cytofectms), DNA-protem complexes, and biopolymers. In addition to a nucleic acid, a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

A "cloning vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bπng about the replication of the attached segment. Cloning vectors may be capable of replication in one cell type, and expression in another ("shuttle vector").

A "cassette" refers to a segment of DNA that can be inserted into a vector at specific restriction sites. The segment of DNA encodes a polypeptide of interest, and the cassette and restπction sites are designed to ensure insertion of the cassette m the proper reading frame for transcπption and translation. A cell has been "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been "transformed" by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change. The transforming DNA can be integrated (covalently hnked) into chromosomal DNA making up the genome of the cell.

A "nucleic acid molecule" refers to the phosphate ester polymeric form of πbonucleosides (adenosme, guanosme, uπdme or cytidine; "RNA molecules") or deoxyπbonucleosides (deoxyadenosme, deoxyguanosme, deoxythymidme, or deoxycytidine; "DNA molecules"), or any phosphoester anologs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, mter aha, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be descπbed herein according to the normal convention of giving only the sequence in the 5 ' to 3 ' direction along the nontranscπbed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropπate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stπngency hybπdization conditions, corresponding to a T_m of 55°, can be used, e.g., 5x SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher T_m, e.g., 40% formamide, with 5x or 6x SCC. High stπngency hybridization conditions correspond to the highest T_m, e.g., 50% formamide, 5x or 6x SCC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stπngency of the hybridization, mismatches between bases are possible. The appropriate stπngency for hybπdizmg nucleic acids depends on the length of the nucleic acids and the degree of complementation, vaπables well known m the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybπds of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_m have been deπved (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e., ohgonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably a minimum length for a hybπdizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.

In a specific embodiment, the term "standard hybridization conditions" refers to a T_m of 55°C, and utilizes conditions as set forth above. In a preferred embodiment, the T_m is 60°C; in a more preferred embodiment, the T_m is 65°C.

As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 18 nucleotides, that is hybπdizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding Akt. Ohgonucleotides can be labeled, e.g., with ³²P-nucleotιdes or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid encoding Akt. In another embodiment, ohgonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of Akt, or to detect the presence of nucleic acids encoding Akt. In a further embodiment, an oligonucleotide of the invention can form a tπple helix with an Akt DNA molecule. Generally, ohgonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, ohgonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated mto a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3 ' to the coding sequence.

Transcπptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3 ' terminus by the transcription initiation site and extends upstream (5 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Withm the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

A coding sequence is "under the control" of transcπptional and translational control sequences m a cell when RNA polymerase transcribes the coding sequence mto mRNA, which is then trans-RNA spliced (if the coding sequence contains mtrons) and translated into the protein encoded by the coding sequence.

As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamihes (e.g., the lmmunoglobuhn superfamily) and homologous proteins from different species (e g , myosm light chain, etc.) (Reeck et al., 1987, Cell 50:667). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similaπty.

Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or ammo acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and not a common evolutionary origin.

In a specific embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by compaπng the sequences using standard software available in sequence data banks, or m a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropπate hybridization conditions is within the skill of the art. See, e.g., Mamatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybπdization, supra.

An "antisense nucleic acid" is a sequence of nucleotides that is complementary to the sense sequence. Antisense nucleic acids can be used to down regulate or block the expression of the polypeptide encoded by the sense strand.

Transcπptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence m a host cell. In eukaryotic cells, polyadenylation signals are additional types of control sequences.

A "signal sequence" is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-termmal to the mature polypeptide, that directs the host cell to translocate the polypeptide. The term "translocation signal sequence" is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.

"Regulatory region" means a nucleic acid sequence which regulates the expression of a second nucleic acid sequence. A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin which are responsible for expressing different proteins or even synthetic proteins (a heterologous region). In particular, the sequences can be sequences of eukaryotic or viral genes or deπved sequences which stimulate or repress transcription of a gene m a specific or non-specific manner and m an mducible or non-mducible manner. Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcπptional termination sequences, signal sequences which direct the polypeptide mto the secretory pathways of the target cell, and promoters.

A regulatory region from a "heterologous source" is a regulatory region which is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur m nature, but which are designed by one having ordinary skill m the art.

"Heterologous" DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell.

"Homologous recombination" refers to the insertion of a foreign DNA sequence into another DNA molecule, e.g., insertion of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.

A "polypeptide" is a polymeric compound comprised of covalently linked ammo acid residues. Ammo acids have the following general structure:

H

R-C-COOH

I

NH₂ Ammo acids are classified mto seven groups on the basis of the side chain R: (1) aliphatic side chams, (2) side chains containing a hydroxyhc (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) prolme, an imino acid in which the side chain is fused to the ammo group. A polypeptide of the invention preferably comprises at least about 14 ammo acids.

A "protein" is a polypeptide which plays a structural or functional role in a living cell.

A "variant" of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants may be allehc vaπations characterized by differences m the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification. The skilled artisan can produce vaπants having single or multiple ammo acid substitutions, deletions, additions, or replacements. These variants may include, inter aha: (a) variants m which one or more ammo acid residues are substituted with conservative or non-conservative ammo acids, (b) variants in which one or more ammo acids are added to the polypeptide or protein, (c) variants in which one or more of the ammo acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.

If such allehc variations, analogues, fragments, deπvatives, mutants, and modifications, including alternative mRNA splicing forms and alternative post-translational modification forms result m deπvatives of the polypeptide which retain any of the biological properties of the polypeptide, they are intended to be included withm the scope of this invention.

A "heterologous protein" refers to a protein not naturally produced in the cell.

Two amino acid sequences are "substantially homologous" or "substantially similar" when greater than about 40% of the ammo acids are identical, or greater than 60% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program.

The term "corresponding to" is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similaπty or homology is measured. A nucleic acid or ammo acid sequence alignment may include spaces. Thus, the term "corresponding to" refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases. Genes Encoding Akt Proteins

The present invention contemplates the use of an Akt protein or polypeptide, or a nucleic acid encoding an Akt protein or polypeptide to stimulate expression of VEGF in cells. Preferably, the Akt is a human Akt3 protein or polypeptide, including a full length, or naturally occurring form of Akt, or any fragment thereof capable of stimulating expression of VEGF. As used herein, "Akt" refers to Akt polypeptide, and "akt" refers to a gene encoding an Akt polypeptide.

Various mouse and human Akt sequences are known in the art (see Coffer et al., 1991, Eur. J. Biochem. 201.475-481; Jones et al., 1991. Proc. Natl. Acad. Sci. 88:4171-4175; Bellacosa et al., 1993, Oncogene, 8:745-754; GenBank Accession Nos. M63167, X61037 and X65687; and US Provisional apphcation number 60/125,108). Preferably, the Akt is human Aktl (SEQ ID NO:l 1), Akt2 (SEQ ID NO: 12) or Akt3 (SEQ ED NO:2) A preferred Akt according to the invention compπses an am o acid sequence as shown m SEQ ID NO: 2. A preferred nucleic acid according to the invention encodes an ammo acid sequence as shown in SEQ ID NO. 2, SEQ ID NO: 11 or SEQ ID NO: 12. More preferably, the nucleic acid compπses a sequence as depicted in SEQ ID NO: 1. The Akt can also be derived from a non-human source.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques withm the skill of the art. Such techniques are explained fully m the literature. See, e.g , Sambrook, Fπtsch & Mamatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgms eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

A gene encoding an Akt, whether genomic DNA or cDNA, can be isolated from any source, particularly from a human cDNA or genomic library. General methods for obtaining an akt gene are well known in the art, as described above (see, e g , Sambrook et al., 1989, supra).

Accordingly, any animal cell potentially can serve as the nucleic acid source for the molecular cloning of a akt gene. The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g , a DNA "library"), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein (e.g., heart, pancreas and skeletal muscle cDNA), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, puπfied from the desired cell (See, for example, Sambrook et al., 1989, supra; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd , Oxford, U.K Vol. I, II) . Clones derived from genomic DNA may contain regulatory and intron DNA regions m addition to coding regions; clones derived from cDNA will not contain mtron sequences Whatever the source, the gene should be molecularly cloned mto a suitable vector for propagation of the gene

Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired akt gene may be accomplished a number of ways. For example, DNA fragments may be screened by nucleic acid hybridization to a labeled probe (Benton and Davis, 1977, Science 196' 180; Grunstem and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybπdize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used. In a specific embodiment, Northern hybridization conditions are used to identify mRNA splicing variants of an akt gene.

Further selection can be carried out on the basis of the properties of the gene, e.g. , if the gene encodes a protein product having the isoelectric, electrophoretic, ammo acid composition, or partial amino acid sequence of Akt protein as disclosed herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, or lmmunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, e g , has similar or identical electrophoretic migration, isoelectric focusing or non-equihbπum pH gel electrophoresis behavior, proteolytic digestion maps, or antigenic properties as known for Akt In a specific embodiment, the expressed protein is recognized by a polyclonal antibody that is generated against an epitope specific for human Akt.

The present invention also relates to the use of genes (e g. , cDNAs) encoding allehc vaπants, splicing variants, analogs, and derivatives of Akt, that have the ability to stimulate the expression of a VEGF. The production and use of Akt derivatives and analogs are withm the scope of the present invention. Such vaπants, analogs, derivatives and homologs should retain the ability to stimulate expression of a VEGF.

Akt deπvatives can be made by alteπng encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Preferably, deπvatives are made that have enhanced or increased functional activity relative to native Akt.

Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same ammo acid sequence as a akt gene, including an ammo acid sequence that contains a single ammo acid variant, may be used in the practice of the present invention. These include but are not limited to allehc genes, homologous genes from other species, and nucleotide sequences comprising all or portions of akt genes which are altered by the substitution of different codons that encode the same ammo acid residue withm the sequence, thus producing a silent change Likewise, the Akt derivatives of the invention include, but are not limited to, those containing, as a primary ammo acid sequence, all or part of the ammo acid sequence of a Akt protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues withm the sequence resulting in a conservative ammo acid substitution For example, one or more ammo acid residues withm the sequence can be substituted by another ammo acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration Substitutes for an ammo acid withm the sequence may be selected from other members of the class to which the ammo acid belongs. For example, the nonpolar (hydrophobic) ammo acids include alanme, leuc e, lsoleucme, valme, prolme, phenylalanme, tryptophan and methionme Ammo acids containing aromatic ring structures are phenylalanme, tryptophan, and tyrosme The polar neutral ammo acids include glycme, serme, threonme, cysteme, tyrosme, asparagme, and glutamme The positively charged (basic) ammo acids include arginine, lysme and histidme The negatively charged (acidic) ammo acids include aspartic acid and glutamic acid Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point Particularly preferred substitutions are-

- Lys for Arg and vice versa such that a positive charge may be maintained,

- Glu for Asp and vice versa such that a negative charge may be maintained,

- Ser for Thr such that a free -OH can be maintained, and

- Gin for Asn such that a free CONH₂ can be maintained

Ammo acid substitutions may also be introduced to substitute an ammo acid with a particularly preferable property. For example, a Cys may be introduced in order to add a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i e , His can act as an acid or base and is the most common ammo acid m biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces β-turns in the protein's structure

The genes encoding Akt derivatives and analogs of the invention can be produced by various methods known m the art The manipulations which result in their production can occur at the gene or protein level For example, the cloned Akt gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and hgated in vitro. In the production of the gene encoding a deπvative or analog of Akt, care should be taken to ensure that the modified gene remains within the same translational reading frame as the Akt gene, uninterrupted by translational stop signals, m the gene region where the desired activity is encoded.

Additionally, the Akt-encodmg nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Preferably, such mutations enhance the functional activity of the mutated Akt gene product Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchmson, C, et al., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA 3:479-488; Ohphant et al., 1986, Gene 44:177; Hutchmson et al., 1986, Proc Natl. Acad Sci. U.S.A. 83:710), use of TAB® linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology Principles and Applications for DNA Amplification, H. Erhch, ed., Stockton Press, Chapter 6, pp. 61-70).

The identified and isolated gene can then be inserted mto an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coh, bacteπophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid deπvatives, e g , pGEX vectors, pmal-c, pFLAG, etc. The insertion mto a cloning vector can, for example, be accomplished by ligatmg the DNA fragment mto a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present m the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligatmg nucleotide sequences (linkers) onto the DNA termini; these hgated linkers may comprise specific chemically synthesized ohgonucleotides encoding restriction endonuclease recognition sequences. Recombinant molecules can be introduced mto host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E coh, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication m both E coh and Saccharomyces cerevisiae by linking sequences from an E coh plasmid with sequences form the yeast 2μ plasmid.

Expression of Akt Polypeptides

The nucleotide sequence coding for Akt, or antigemc fragment, derivative or analog thereof, or a functionally active deπvative, including a chimeπc protein, thereof, can be inserted mto an appropriate expression vector, ι . , a vector which contains the necessary elements for the transcription and translation of the inserted protem-codmg sequence. Such elements are termed herein a "promoter." Thus, the nucleic acid of the invention is operationally associated with a promoter in an expression vector of the invention Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. An expression vector also preferably includes a replication origin.

The necessary transcπptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding the Akt and/or its flanking regions. In one preferred embodiment, the expression of Akt is restricted to cardiomyocytes using a cardiac specific promoter and/or a vector with specific tropism for cardiac cells.

Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteπophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcπption and translation elements may be used.

A recombinant Akt protein, or functional fragment, derivative, chimeπc construct, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).

A cell containing a recombinant vector compπsmg the nucleic acid encoding an Akt may be cultured m an appropriate cell culture medium under conditions that provide for expression of Akt by the cell. Any of the methods previously described for the insertion of DNA fragments mto a cloning vector may be used to construct expression vectors containing a gene consisting of appropπate transcπptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).

A nucleic acid encoding an Akt polypeptide may be operably linked and controlled by any regulatory region, i.e., promoter/enhancer element known in the art, but these regulatory elements must be functional in the host target tumor selected for expression. The regulatory regions may comprise a promoter region for functional transcription in the host cell, as well as a region situated 3^* of the gene of interest, and which specifies a signal for termination of transcπption and a polyadenylation site. All these elements constitute an expression cassette. Promoters that may be used in the present invention include both constitutive promoters and regulated (mducible) promoters The promoter may be naturally responsible for the expression of the nucleic acid. It may also be from a heterologous source. In particular, it may be promoter sequences of eukaryotic or viral genes. For example, it may be promoter sequences derived from the genome of the cell which it is desired to mfect. Likewise, it may be promoter sequences derived from the genome of a virus, such as adenovirus (E1A and MLP), cytomegalovirus, or Rous Sarcoma Virus. In addition, the promoter may be modified by addition of activating or regulatory sequences or sequences allowing a tissue-specific or predominant expression (enolase and GFAP promoters and the like). Moreover, when the nucleic acid does not contain promoter sequences, it may be inserted.

Some promoters useful for practice of this invention are ubiquitous promoters (e g , HPRT, vimentm, actm, tubuhn), intermediate filament promoters (e g , desmm, neurofilaments, keratm, GFAP), therapeutic gene promoters (e g , MDR type, CFTR, factor VIE), tissue-specific promoters (e g , actm promoter in smooth muscle cells), promoters which are preferentially activated m dividing cells, promoters which respond to a stimulus (e g , steroid hormone receptor, retmoic acid receptor), tetracyclme -regulated transcπptional modulators, cytomegalovirus (CMV) immediate-early, retroviral LTR, metallothionem, SV-40, adenovirus Ela, and adenovirus major late (MLP) promoters. Tetracyclme-regulated franscriptional modulators and CMV promoters are described m WO 96/01313, US 5,168,062 and 5,385,839, the contents of which are incorporated herein by reference.

More specifically, expression of Akt protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787- 797), the herpes thymidme kmase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445), the regulatory sequences of the metallothionem gene (Bπnster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kmase) promoter, alkaline phosphatase promoter; and the animal transcπptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spπng Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122), immunoglobulm gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7.1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1 :268-276), alpha-fetoprotem gene control region which is active m liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1-antιtrypsm gene control region which is active m the liver (Kelsey et al., 1987, Genes and Devel. 1 : 161-171), beta-globm gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kolhas et al., 1986, Cell 46:89-94), myelm basic protein gene control region which is active m ohgodendrocyte cells m the bram (Readhead et al., 1987, Cell 48:703-712), myosm light chaιn-2 gene control region which is active in skeletal muscle (Sam, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Preferably, expression of Akt is restricted to cardiomyocytes using either a cardiac specific promoter, or a vector with a specific tropism for cardiac cells.

Expression vectors containing a nucleic acid encoding a Akt protein can be identified by five general approaches: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection marker gene functions, (d) analyses with appropriate restriction endonucleases, and (e) expression of inserted sequences. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybπdization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g., β-galactosidase activity, thymidme kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. In another example, if the nucleic acid encoding Akt is inserted within the "selection marker" gene sequence of the vector, recombmants containing the Akt insert can be identified by the absence of the gene function. In the fourth approach, recombinant expression vectors are identified by digestion with appropriate restπction enzymes. In the fifth approach, recombinant expression vectors can be identified by assaying for the activity, biochemical, or lmmunological characteπstics of the gene product expressed by the recombinant, provided that the expressed protein assumes a functionally active conformation. A wide variety of host/expression vector combinations may be employed in expressing Akt DNA sequences. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coh plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al , 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2m plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful m insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

For example, in a baculovirus expression systems, both non-fusion transfer vectors, such as but not limited to pVL941 (BamHl cloning site; Summers), pVL1393 (_3αmHl, Smal, Xbal, EcoRI, Notl, Xmalll, Bglϊ , and Pstl cloning site; Invifrogen), pVL1392 (Bglll, Pstl, Notl, Xma R, EcoRI, Xbal, Smal, and BamHl cloning site; Summers and Invifrogen), and pBlue_9_.cIH (BamHl, BglE, Pstl, Ncol, and Hindϊil cloning site, with blue/white recombinant screening possible; Invifrogen), and fusion transfer vectors, such as but not limited to pAc700 (BamHl and Kpnl cloning site, m which the BamHl recognition site begins with the initiation codon; Summers), pAc701 and pAc702 (same as pAc700, with different reading frames), pAc360 (BamHl cloning site 36 base pairs downstream of a polyhedrm initiation codon; Invιtrogen(195)), and pBlueBacHisA, B, C (three different reading frames, with BamHl, BgHl, Pstl, Ncol, and Hindm cloning site, an N-terminal peptide for ProBond puπfication, and blue/white recombinant screening of plaques; Invitrogen (220)) can be used.

Mammalian expression vectors contemplated for use the invention include vectors with mducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e g. , any expression vector with a DHFR expression vector, or a DHER/methotrexate co-amplification vector, such as pΕD (Pstl, Sail, Sbal, Smal, and EcoRI cloning site, with the vector expressing both the cloned gene and DHFR; see Kaufman, Current Protocols in Molecular Biology, 16.12 (1991). Alternatively, a glutamme synthetase/methionme sulfoximme co-amplification vector, such as pΕΕ14 (HindΩI, Xbal, Smal, Sbal, EcoRI, and Bell cloning site, in which the vector expresses glutamme synthase and the cloned gene; Celltech). In another embodiment, a vector that directs episomal expression under control of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamHl, Sfil, Xhol, Notl, Nhel, HindRl, Nhel, Ev_.II, and Kpnl cloning site, constitutive Rous Sarcoma Virus Long Terminal Repeat (RSV-LTR) promoter, hygromycm selectable marker; Invitrogen), pCΕP4 (BamHl, Sfil, Xhol, Notl, Nhel, HindΩI, Nhel, Ev_.II, and Kpnl cloning site, constitutive human cytomegalovirus (hCMV) immediate early gene, hygromycm selectable marker; Invitrogen), pMEP4 (Kpnl, Pvul, Nhel, HindHl, Λfotl, Xhol, Sfil, BamHl cloning site, mducible methallothione Ha gene promoter, hygromycm selectable marker: Invitrogen), pREP8 (BamHl, Xhol, Notl, HindEΪ, Nhel, and Kpnl cloning site, RSV-LTR promoter, histidmol selectable marker; Invitrogen), pREP9 (Kpnl, Nhel, Hindϊll, Notl, Xhol, Sfil, and BamHl cloning site, RSV-LTR promoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycm selectable marker, N-termmal peptide puπfiable via ProBond resm and cleaved by enterokmase; Invitrogen). Selectable mammalian expression vectors for use m the invention include pRc/CMV (HindH , BstXl, Notl, Sbal, and Apal cloning site, G418 selection; Invitrogen), pRc/RSV (HindΩl, Spel, BstXl, Notl, Xbal cloning site, G418 selection, Invitrogen), and others. Vaccinia virus mammalian expression vectors (see, Kaufman, 1991, supra) for use according to the invention include but are not limited to pSC 1 1 (Smal cloning site, TK- and β- gal selection), pMJ601 (Sail, Smal, Afil, Narl, BspMll, BamHl, Apal, Nhel, Sacll, Kpnl, and HindlU cloning site. TK- and β-gal selection), and pTKgptFIS (EcoRI, Pstl, Sail, Accl, Hindu, Sbal, BamHl, and Hpa cloning site, TK or XPRT selection).

Yeast expression systems can also be used according to the invention to express Akt protein For example, the non-fusion pYΕS2 vector (Xbal, Sphl, Shol, Notl, GstXl, EcoRI, BstXl, BamHl, Sad, Kpnl, and HindΩl cloning sit; Invitrogen) or the fusion pYESHisA, B, C (Xbal, Sphl, Shol, Notl, BstXl, EcoRI, BamHl, Sad, Kpnl, and HindHl cloning site, N-termmal peptide purified with ProBond resm and cleaved with enterokmase; Invitrogen), to mention just two, can be employed according to the invention.

Once a particular recombmant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their deπvatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteπophage vectors (e g , lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product m the specific fashion desired. Different host cells have characteπstic and specific mechanisms for the translational and post-translational processing and modification of proteins. Appropπate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. Expression in yeast can produce a biologically active product. Expression m eukaryotic cells can increase the likelihood of "native" folding. Moreover, expression in mammalian cells can provide a tool for reconstituting, or constituting, Akt activity. Furthermore, different vector/host expression systems may affect processmg reactions, such as proteolytic cleavages, to a different extent.

Vectors are introduced mto the desired host cells by methods known in the art, e g , transfection, electroporation, micromjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, hpofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e g , Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621- 14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).

Soluble forms of the protein can be obtained by collecting culture fluid, or solubihzing inclusion bodies, e g , by treatment with detergent, and if desired somcation or other mechanical processes, as described above. The solubihzed or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dιmensιonal gel electrophoresis, chromatography (e.g , ion exchange, affinity, lmmunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.

Gene Therapy and Transgenic Vectors

As discussed above, the present invention relates to the ability of Akt proteins to stimulate expression of VEGF, a protein that induces angiogenesis. Therefore, the present invention includes gene therapy by the administration of a nucleic acid encoding an Akt protein to a patient suffering from an ischemic condition. Ischemic conditions may include cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, peripheral arteπal disease, intermittent claudication, myocardial ischemia, or ischemic, idiopathic or hypertrophic cardiomyopathy.

A nucleic acid encoding Akt, where appropriate incorporated in vectors, and the pharmaceutical compositions containing them, may be used for the treatment of ischemic tissue. They may be used for the transfer and expression of genes in vivo in any type of ischemic tissue, especially the heart. The treatment can, moreover, be targeted m accordance with the pathology to be treated (transfer to a particular tissue can, in particular, be determined by the choice of a vector, and expression by the choice of a particular promoter) The nucleic acids or vectors of the invention are advantageously used for the production in humans or animals, in vivo and mtracellularly, of Akt proteins capable of stimulating the expression of VEGF proteins. The present invention thus makes it possible to treat specifically, locally and effectively ischemia.

A nucleic acid encoding an Akt protein can be administered alone or m combination with a nucleic acid encoding an angiogenic factor. Known angiogenic factors include basic and acidic fibroblast growth factors (bFGF and aFGF), FGF-5 (US Pat. 5,661,133), endothehal cell growth factor (Pu et al., 1993, Circulation 88:208-2156), angiopoietm and VEGF (for reviews see Mehllo et al., 1997 and Lewis et al., 1997). Several forms of VEGF have been identified, including VEGF₁₂₁ (US Pat. 5,219,739), VEGF,₆₅ (US Pat. 5,332,672), VEGF₁₈₉ (US Pat 5.240,848), VEGF₂₀₆, VEGF-2 (W095/24473; W096/39515), VEGF-B (US Pats. 5,607,918 and 5,840,693), and VEGF-D (W097/12972). A nucleic acid encoding an Akt protein can also be administered in combination with a transition metal ion, such as CoCl₂, which has been shown to enhance expression of the VEGF gene and to stimulate vasculaπzation (US. Pat. 5,480,975).

As discussed above, a "vector" is any means for the transfer of a nucleic acid according to the invention mto a host cell Preferred vectors are viral vectors, such as retroviruses, herpes viruses, adenoviruses, and adeno-associated viruses. Thus, a gene encoding an Akt protein or polypeptide is introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA Expression m targeted tissues can be effected by targeting the transgemc vector to specific cells, such as with a viral vector or a receptor hgand, or by using a tissue-specific promoter, or both.

Viral vectors commonly used for in vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art [see, e.g , Miller and Rosman, BioTechmques 7:980-990 (1992)]. Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors which are used withm the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled m the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagemc agents. Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsulating the viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), vaccinia virus, and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not replication competent after introduction into a cell, and thus does not lead to a productive viral infection. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector [Kaphtt et al., Molec. Cell Neurosci. 2:320-330 (1991)], defective herpes virus vector lacking a glyco-protein L gene [Patent Publication RD 371005 A], or other defective herpes virus vectors [International Patent Publication No WO 94/21807, published September 29, 1994: International Patent Publication No. WO 92/05263, published April 2, 1994]; an attenuated adenovirus vector, such as the vector described by Stratford- Perπcaudet et al. [J Chn Invest 90:626-630 (1992); see also La Salle et al., Science 259:988-990 (1993)]; and a defective adeno-associated virus vector [Samulski et al., J. Virol 6L3096-3101 (1987); Samulski et al., J Virol 63:3822-3828 (1989); Lebkowski et al., Mol Cell Biol 8:3988-3996 (1988)].

Preferably, for in vivo administration, an appropriate lmmunosuppressive treatment is employed m conjunction with the viral vector, e g., adenovirus vector, to avoid lmmuno-deactivation of the viral vector and transfected cells. For example, lmmunosuppressive cytokmes, such as mterleukm-12 (IL-12), mterferon-γ (IFN-γ), or antι-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors [see, e g , Wilson, Nature Medicine (1995)]. In addition, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens

Naturally, the invention contemplates delivery of a vector that will express a therapeutically effective amount of Akt for gene therapy applications. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant ischemic condition in a host.

Adenovirus vectors

In a preferred embodiment, the vector is an adenovirus vector. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Various serotypes of adenovirus exist. Of these serotypes, preference is given, withm the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal oπgm (see W094/26914). Those adenoviruses of animal origin which can be used withm the scope of the present invention include adenoviruses of canme, bovme, muπne (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian, and simian (example: SAV) oπgm. Preferably, the adenovirus of animal oπgm is a canme adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800), for example).

Preferably, the replication defective adeno viral vectors of the invention comprise the ITRs, an encapsidation sequence and the nucleic acid of interest. Still more preferably, at least the El region of the adenoviral vector is non-functional. The deletion in the El region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (PvuII-Bglll fragment) or 382 to 3446 (HmfII-Sau3A fragment). Other regions may also be modified, in particular the E3 region (WO95/02697), the E2 region (W094/28938), the E4 region (W094/28152, W094/12649 and WO95/02697), or in any of the late genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in the El region (Ad 1.0). Examples of El-deleted adenoviruses are disclosed in EP 185,573, the contents of which are incorporated herein by reference. In another preferred embodiment, the adenoviral vector has a deletion in the El and E4 regions (Ad 3.0). Examples of El/E4-deleted adenoviruses are disclosed m WO95/02697 and W096/22378, the contents of which are incorporated herein by reference. In still another preferred embodiment, the adenoviral vector has a deletion in the El region mto which the E4 region and the nucleic acid sequence are inserted (see FR94 13355, the contents of which are incorporated herein by reference).

The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid which carries, inter aha, the DNA sequence of interest. The homologous recombination is effected following cotransfection of the adenovirus and plasmid into an appropπate cell line. The cell line which is employed should preferably (I) be transformable by the said elements, and (n) contain the sequences which are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination. Examples of cell lines which may be used are the human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines which are able to complement the El and E4 functions, as described m applications W094/26914 and WO95/02697. Recombinant adenoviruses are recovered and puπfied using standard molecular biological techniques, which are well known to one of ordinary skill in the art.

Adeno-associated virus vectors

The adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved m human pathologies. The AAV genome has been cloned, sequenced and characterised. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an oπgin of replication for the virus. The remainder of the genome is divided into two essential regions which carry the encapsulation functions: the left-hand part of the genome, which contains the rep gene involved in viral replication and expression of the viral genes; and the πght-hand part of the genome, which contains the cap gene encoding the capsid proteins of the virus. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488 528). These publications descπbe various AAV-deπved constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferπng the said gene of interest in vitro (mto cultured cells) or in vivo, (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by cotransfectmg a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsulation genes (rep and cap genes), mto a cell line which is infected with a human helper virus (for example an adenovirus). The AAV recombmants which are produced are then purified by standard techniques.

The invention also relates, therefore, to an AAV-deπved recombinant virus whose genome encompasses a sequence encoding a nucleic acid encoding an Akt3 flanked by the AAV ITRs. The invention also relates to a plasmid encompassing a sequence encoding a nucleic acid encoding an Akt3 flanked by two ITRs from an AAV. Such a plasmid can be used as it is for transferring the nucleic acid sequence, with the plasmid, where appropriate, being incorporated mto a liposomal vector (pseudo-virus).

Retrovirus vectors

In another embodiment the gene can be introduced in a retroviral vector, e.g. , as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U.S. Patent No. 4,650,764; Temm et al., U.S. Patent No. 4,980,289; Markowitz et al., 1988, J. Virol. 62:1120; Temm et al., U.S. Patent No. 5,124,263; EP 453242, EP178220; Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689; International Patent Publication No. WO 95/07358, published March 16, 1995, by Dougherty et al.; and Kuo et al., 1993, Blood 82:845. The retroviruses are integrating viruses which infect dividing cells. The retrovirus genome includes two LTRs, an encapsulation sequence and three coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and e«v genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("muπne Moloney leukaemia virus" MSV ("murme Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Defective retroviral vectors are disclosed in WO95/02697.

In general, in order to construct recombinant retroviruses containing a nucleic acid sequence, a plasmid is constructed which contains the LTRs, the encapsulation sequence and the coding sequence. This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes Such packaging cell lines have been described in the prior art, m particular the cell line PA317 (US4,861,719); the PsiCRIP cell line (WO90/02806) and the GP⁺envAm^"12 cell line (WO89/07150). In addition, the recombinant retroviral vectors can contain modifications withm the LTRs for suppressing transcπptional activity as well as extensive encapsulation sequences which may include a part of the gag gene (Bender et al , J. Virol. 61 (1987) 1639) Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

Retroviral vectors can be constructed to function as infections particles or to undergo a single round of transfection In the former case, the virus is modified to retain all of its genes except for those responsible for oncogemc transformation properties, and to express the heterologous gene Non- mfectious viral vectors are prepared to destroy the viral packaging signal, but retain the structural genes required to package the co-introduced virus engineered to contain the heterologous gene and the packaging signals Thus, the viral particles that are produced are not capable of producing additional virus.

Targeted gene delivery is described m International Patent Publication WO 95/28494, published October 1995.

Non-viral vectors

Alternatively, the vector can be introduced in vivo by hpofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro Synthetic catiomc hpids designed to limit the difficulties and dangers encountered with hposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner, et. al., Proc Natl Acad. Sci USA 84-7413-7417 (1987); see Mackey, et al., Proc Natl Acad Sci USA 85:8027-8031 (1988); Ulmer et al , Science 259:1745-1748 (1993)]. The use of catiomc hpids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes [Feigner and Rmgold, Science 337.387-388 (1989)]. Particularly useful hpid compounds and compositions for transfer of nucleic acids are described in International Patent Publications W095/18863 and W096/17823, and m U.S. Patent No. 5,459,127 The use of hpofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit It is clear that directing transfection to particular cell types would be particularly advantageous m a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the bram. Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, et. al., supra]. Targeted peptides, e g , hormones or neurotransmitters, and proteins such as antibodies, or non- peptide molecules could be coupled to liposomes chemically. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a catiomc ohgopeptide (e g , International Patent Publication W095/21931), peptides derived from DNA binding proteins (e g , International Patent Publication WO96/25508), or a catiomc polymer (e g , International Patent Publication W095/21931)

It is also possible to introduce the vector in vivo as a naked DNA plasmid (see U S Patents 5,693,622, 5,589,466 and 5,580,859) Naked DNA vectors for gene therapy can be introduced mto the desired host cells by methods known in the art, e g , transfection, electroporation, micromjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, e g , Wu et al , J Biol Chem 267 963-967 (1992), Wu and Wu, J Biol Chem 263 14621-14624 (1988), Hartmut et al , Canadian Patent Application No 2,012,311, filed March 15, 1990, Williams et al , Proc Natl Acad Sci USA 88 2726-2730 (1991)] Receptor- mediated DNA delivery approaches can also be used [Cuπel et al , Hum Gene Ther 3 147-154 (1992), Wu and Wu, J Biol Chem 262 4429-4432 (1987)] Preferred naked DNA vectors include pCOR plasmids having a conditional origin of replication (see WO97/10343), and mmicircle plasmids lacking an origin of replication and a marker gene (see WO96/26270)

Pharmaceutical Compositions and Delivery

The present invention also relates to a pharmaceutical compositions Such compositions may comprise an Akt protein or polypeptide or a nucleic acid encoding an Akt protein or polypeptide, as defined above, and a pharmaceutically acceptable earner or vehicle The compositions of the invention are particularly suitable for formulation of biological mateπal for gene therapy Thus, in a preferred embodiment, the composition comprises a nucleic acid encoding a human Akt protein or polypeptide

The composition may comprise an Akt protein, or nucleic acid encoding an Akt protein The composition may, in addition, comprise a nucleic acid encoding an angiogenic factor Known angiogenic factors include basic and acidic fibroblast growth factors (bFGF and aFGF), FGF-5 (US Pat 5,661,133), endothehal cell growth factor (Pu et al , 1993, Circulation 88 208-2156), angiopoietm and VEGF (for reviews see Mehllo et al , 1997 and Lewis et al , 1997) Nucleic acids encoding several forms of VEGF have been identified, including VEGF₁₂₁ (US Pat 5,219,739), VEGF₁₆₅ (US Pat 5,332,672), VEGF₁₈₉ (US Pat 5,240,848), VEGF₂₀₆, VEGF-2 (W095/24473, W096/39515), VEGF-B (US Pats 5,607,918 and 5,840,693), and VEGF-D (W097/12972) The composition may also compπse a transition metal ion, such as CoCl₂, which has been shown to enhance expression of the VEGF gene and to stimulate vasculaπzation (US Pat 5,480,975) The Akt protein, or nucleic acid encoding an Akt protein may also be administered m conjunction with a vasodilator. Examples of vasodilators include mtrovasodilators (e.g. mtroprusside, nitroglyceπn), non-specific vasodilators (e.g. hyrdrahzine, papaverme), adenosme receptor agonists, calcium channel blocking agents, alpha blockers (e.g. prazosm), endogenous vasodilator peptides or related peptide analogs (e.g. substance P, CGRP), K channel activators, ACE inhibitors or angiotensin receptor blockers, endothelm receptor blockers or ECE inhibitors, and vasodilator prostaglandms.

Any vector, viral or non-viral, of the invention will preferably be introduced in vivo in a pharmaceutically acceptable vehicle or carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution salme solutions and aqueous dextrose and glycerol solutions are preferably employed as earners, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

The pharmaceutical compositions of the invention may be formulated for the purpose of topical, oral, parenteral, mtranasal, intravenous, intramuscular, subcutaneous, intraocular, and the like, administration.

Preferably, the pharmaceutical compositions contain pharmaceutically acceptable vehicles for an injectable formulation. These can be, m particular, sterile, isotomc salme solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, and the like, or mixtures of such salts), or dry, in particular lyophihzed, compositions which, on addition, as appropriate, of stenhzed water or of physiological saline, enable injectable solutions to be formed.

The compositions may be m particular isotomc, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the hke or mixtures of such salts), or dry, especially freeze-dπed compositions which upon addition, depending on the case, of stenhzed water or physiological salme, allow the constitution of injectable solutions.

The preferred stenle injectable preparations can be a solution or suspension in a nontoxic parenterally acceptable solvent or diluent. Examples of pharmaceutically acceptable earners or vehicles are salme, buffered salme, isotomc salme (e.g., monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof. 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glyceπdes. Fatty acids such as oleic acid also find use in the preparation of mjectables.

The doses of nucleic acids of the invention, either alone or incorporated m a vector, used for administration can be adjusted in accordance with different parameters, and in particular in accordance with the mode of administration used, the pathology in question, the gene to be expressed or the desired treatment period. Generally speaking, m the case of the recombinant viruses according to the invention, these are formulated and administered m the form of doses of between 10⁴ and 10¹⁴ pfu, and preferably 10⁶ to 10¹⁰ pfu. The term pfu (plaque forming unit) corresponds to the infectious power of a solution of virus, and is determined by infection of a suitable cell culture and measurement, generally after 48 hours, of the number of infected cell plaques. The techniques of determination of the pfu titre of a viral solution are well documented in the literature.

The composition of the invention may be introduced parenterally or transmucosally, e.g., orally, nasally, or rectally, or transdermally. Preferably, administration is parenteral, e.g., via intravenous injection, and also including, but is not limited to, mtra-arteπole, intramuscular, mtradermal, subcutaneous, mtrapeπtoneal, mtraventπcular, and mtracramal administration. The administration of the composition may introduced by injection directly into the site to be treated, in particular, mto the heart.

The preferred route of administration to the heart is by direct injection mto the heart (US Pats. 5,693,622 or 5,661,133). The heart can be imaged using any of the techniques available m the art, such as magnetic resonance imaging or computer-assisted tomography, and the therapeutic composition administered by stereotactic injection, for example, mto ischemic regions of the myocardium.

Preferably, expression of Akt is restncted to cardiomyocytes using either a cardiac specific promoter, or a vector with a specific tropism for cardiac cells.

Administration to the heart can also occur through the use of a catheter. Vanous porous balloon, double balloon and hydrogel catheters are described m US patents 5,851,521, 5,652,225, 5,328,470, 5,698,531, 5,707,969, 5,830,879, and 5,674,192.

In yet another embodiment, a composition compnsmg an Akt polypeptide. or nucleic acid encoding the polypeptide, can be delivered in a controlled release system. For example, the nucleic acid or polypeptide may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used [see Langer, supra; Sefton, CRC Crit Ref Biomed Eng 14:201 (1987); Buchwald et al , Surgery 88:507 (1980); Saudek et al., N Engl. J Med 321 :574 (1989)]. In another embodiment, polymeric materials can be used [see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida (1974); Controlled Drug Bioavailabihty, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley- New York (1984); Ranger and Peppas, J Macromol Sci Rev Macromol Chem. 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann Neurol 25:351 (1989); Howard et al., J Neurosurg 71 : 105 (1989)]. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i e , the heart, thus requiring only a fraction of the systemic dose [see, e g , Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)]. Other controlled release systems are discussed in the review by Langer [Science 249: 1527-1533 (1990)].

Thus, the compositions of the invention can be delivered by intravenous, mtraarteπal, mtrapentoneal, intramuscular, or subcutaneous routes of administration Alternatively, the compositions, properly formulated, can be administered by nasal or oral administration. A constant supply of the biological material can be ensured by providing a therapeutically effective dose (. e. , a dose effective to induce metabolic changes m a subject) at the necessary intervals, e g , daily, every 12 hours, etc. These parameters will depend on the severity of the disease condition being treated, other actions, such as diet modification, that are implemented, the weight, age, and sex of the subject, and other criteria, which can be readily determined according to standard good medical practice by those of skill m the art.

The present invention also relates to a method of inhibiting angiogenesis in a patient suffeπng from a tumor by inhibiting the level of Akt activity in the patient, thereby inhibiting production of VEGF. The level of Akt may be decreased by introducing an Akt antisense nucleic acid mto cells of the patient under conditions wherem the antisense nucleic acid hybridizes under intracellular conditions to an Akt mRNA. The level of Akt can also be decreased by introducing an intracellular binding protein, such as a single chain Fv antibody (scFv), that specifically binds Akt into a patient's cell at a level sufficient to bind to and inactivate Akt. In another embodiment, Akt activity can be decreased by admimstenng a nucleic acid encoding a dominant negative form of an Akt. Preferably, the antisense nucleic acid, intracellular binding protein or nucleic acid encoding therefor, or dominant negative are administered directly to tumor cells.

An antisense sequence according to the invention is complementary to the sequence encoding an Akt protein and down-regulates or blocks expression of the Akt protein. A preferred embodiment comprises an antisense polynucleotide molecule. Preparation and use of antisense polynucleotides, DNA encoding antisense RNA molecules and use of ohgo and genetic antisense is disclosed in WO 92/15680, the entire contents of which are incorporated herein by reference

Antisense nucleic acids of the invention are preferably RNA capable of specifically hybridizing with all or part of DNA sequence encoding an Akt protein, or the corresponding messenger RNA. The antisense sequence of the present invention may be derived from DNA sequences whose expression m the cell produces RNA complementary to all or part of a human Akt mRNA. These antisense sequences can be prepared by expression of all or part of a sequence encoding an Akt protein in the opposite orientation (EP 140 308) Any length of the antisense sequence is suitable for practice of the invention so long as it is capable of down-regulatmg or blocking expression of the Akt. Preferably, the antisense sequence is at least 20 nucleotides m length

In another aspect of this preferred embodiment the nucleic acid encodes antisense RNA molecules In this embodiment, the nucleic acid is operably hnked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced mto the cell. Examples of suitable vectors includes plasmids, adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses.

A second embodiment of the present invention's method of specifically inhibiting angiogenesis through the inhibition of Akt activity comprises expression of a nucleic acid sequence encoding an intracellular binding protein capable of selectively interacting with the Akt with a transfected cell WO 94/29446 and WO 94/02610, the entire contents of which are incorporated herein by reference, disclose cellular transfection with genes encoding an intracellular binding protein. An intracellular binding protein includes any protein capable of selectively interacting, or binding, with Akt in the cell in which it is expressed and of neutralizing the function of bound Akt protein Preferably, the intracellular binding protein is an antibody or a fragment of an antibody.

WO 94/02610 discloses preparation of antibodies and identification of the nucleic acid encoding a particular antibody. Using an Akt protein, or a fragment thereof, a monoclonal antibody specific for the protein is prepared according to techniques known to those skilled in the art. A vector comprising the nucleic acid encoding an intracellular binding protein, or a portion thereof, and capable of expression in a host cell is subsequently prepared for use in the method of this invention. Suitable vectors and methods of dehveπng nucleic acids encoding intracellular binding proteins to cells containing Akt include those discussed above. The nucleic acid sequence encoding an Akt intracellular binding protein may additionally compπse a sequence encoding a localization signal for targeting the intracellular binding protein to the cellular location of Akt and/or a sequence enabling insertion of the intracellular binding protein mto the plasma membrane. The localization signal or insertion sequence can be located anywhere on the intracellular binding protein, so long as it does not mterfere with binding to the Akt protein. Examples of localization signals are disclosed in WO 94/02610.

In another embodiment, Akt activity can be decreased by administering a nucleic acid encoding a dominant negative form of an Akt. Examples of dominant negative forms of Akt are described m Fujio et al., 1999 (J. Biol. Chem. 274(23): 16349-16354), Wang et al., 1999 (Mol. Cell Biol. 19(6): 4008-4018), Jiang et al., 1999 (Proc. Natl. Acad. Sci. 96(5): 2077-2081), and Gerber et al., 1998 (J. Biol. Chem. 273(46): 30336-30343).

An organism m whom administration of a biological material withm the scope of the invention is administered is preferably a human, but can be any animal. Thus, as can be readily appreciated by one of ordinary skill in the art, the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equme, caprme, ovine, and porcine subjects, wild animals (whether m the wild or a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

The present invention may be better understood by reference to the following non-hmitmg Examples, which are provided as exemplary of the invention.

EXAMPLES

General molecular biology techniques

The methods traditionally used in molecular biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in a caesium chloπde gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, protein extraction with phenol or phenol/chloroform, ethanol or isopropanol precipitation of DNA m a salme medium, transformation in Escheπchia coh, and the like, are well known to a person skilled m the art and are amply described in the literature [Mamatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; (2^nd Ed. 1989); Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].

Conventional cloning vehicles include pBR322 and pUC type plasmids and phages of the Ml 3 seπes. These may be obtained commercially (Bethesda Research Laboratones).

For hgation, DNA fragments may be separated according to their size by agarose or acrylamide gel electrophoresis, extracted with phenol or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA hgase (Biolabs) according to the supplier's recommendations.

The filling in of 5' protruding ends may be performed with the Klenow fragment of E. coh DNA polymerase I (Biolabs) according to the supplier's specifications. The destruction of 3' protruding ends is performed in the presence of phage T4 DNA polymerase (Biolabs) used according to the manufacturer's recommendations. The destruction of 5' protruding ends is performed by a controlled treatment with SI nuclease.

Mutagenesis directed in vitro by synthetic ohgodeoxynucleotides may be performed according to the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.

The enzymatic amplification of DNA fragments by PCR [Polymerase-catalyzed Cham Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mulhs K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] technique may be performed using a "DNA thermal cycler" (Perkm Elmer Cetus) according to the manufacturer's specifications.

Verification of nucleotide sequences may be performed by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

Plasmid DNAs may be purified by the Qiagen Plasmid Purification System according to the manufacture's instruction. Example 1 : Cloning of Human Akt3

This example describes the cloning of a nucleic acid encoding Akt3 protein. Example 1.1 cDNA library screening for Akt 3

A data base search revealed that one human cDNA clone contains a stretch of human cDNA sequence that is homologous to, but different from human Aktl and Akt2. To isolate the full length coding sequence of this previously unknown human Akt lsoform (herein named human Akt3), a human heart cDNA library was screened with a cDNA probe corresponding to the 5'-UTR and coding region for the N-termmus of human Akt3.

A human cDNA clone (ID# 479072) was purchased (Genome System Inc.). One fragment of this DNA, which covers part of the 5'-UTR (untranslated region) and part of the 5 ^'-coding sequence of human Akt3, was amplified by polymerase chain reaction (PCR) using the following primers: AKT3-5'UTR-F3 (5' TCC AAA CCC TAA AGC TGA TAT CAC 3'; SEQ ID NO:3) and AKT3-C- Rl (5' CCT GGA TAG CTT CTG TCC ATT C 3'; SEQ ID NO:4). A cDNA probe was labeled with [α-p32]dCTP using a Random Primer DNA labeling kit (Boerhmger Mannheim) according to the manufacture's instructions. The probe was purified using a Bio-Rad chromatography spin column according to the manufacture^'s instruction. Over one million phage clones were initially used for cDNA phage library screening (Clonetech, Cat# HL5027t). Host cells XL1-B were inoculated at 37°C overnight in LB media (supplemented with 20 mg/ml tetracyclme, 0.2% maltose and lOmM MgC12). Phage infection and membrane lifting were carried out as descπbed in Mamatis, 1989. Membranes were denatured, renatured and baked, then pre-hybridized with hybridization solution for 4 hours at 65°C. A denatured form of the p32-labeled probe (heat denatured for 10 minutes) was added to the membranes for overnight hybridization. After hybridization, membranes were serially washed with 2XSSC/0.1%SDS, 1XSSC/0.1%SDS, and 0.5XSSC/0.1%SDS at 65°C. Membranes were air-dried and exposed to Kodak X-ray films. After this primary screening, positive clones were selected for secondary and tertiary screening. Resulting positive phages were purified, and phage DNA converted mto plasmid DNA using BM25.8-25 host cells according to the manufacture's (Boerhmger Mannheim) instructions.

Two positive clones were chosen for complete sequencing and further characterization. One of these clones (clone #9) comprises part of the 5'-UTR and the N-terminal coding sequence (aa 1 to 127) of human Akt3. A second clone (clone #1) comprises most of the human Akt3 sequence (aa 15 to the C-terminus) and 3'-UTR. A full length cDNA sequence was formed by the fusion of these two partial sequences. A complete sequence encoding a human Akt3 is shown m SEQ ID NO:l. The corresponding ammo acid sequence is shown in SEQ ID NO:2. Akt3 is shorter that Aktl and Akt2, and there is no significant homology between Akt3 and Aktl or Akt2 at the C-termmus of the molecules. In particular, the last 14 ammo acids m the C-termmal portion of human Akt-3 are different from those present m human Aktl and Akt2. Example 2: Construction of Akt3 expression plasmids

This example describes the construction of an expression plasmid for activated Akt3. First two partial cDNA clones (clone #1 and clone #9, described above) were fused to obtain a full length AKT3 coding sequence. A DNA comprising the human Src myπstylation sequence was fused to the N-termmus of the full length Akt3 sequence. An HA-tag sequence was fused to the C-termmus of the full length Akt3 sequence (for detection of expression). The sequence for this chimeπc MyrAkt3HA was placed under the control of a CMV promoter. The complete construct is called CMV6- MyrAkt3HA (Figure 1A). Example 2.1 ^■ CMV6-MyrAktHA

This example describes the construction of plasmids capable of expressing Akt3 and a constitutively active form of human Akt3. A full length Akt3 coding sequence was obtained by PCR amplification of clone #1 using the following primers: hAKT3cl9-PCR5(F): (5'- ATG AGC GAT GTT ACC ATT GTG AAA GAA GGT TGG GTT CAG AAG AGG GGA GAA TAT ATA AAA AAC TGG AGG CCA AG - 3'; SEQ ID NO:5), which contains the coding sequence of the first 24 amino acids of Akt3, and hAKT3 cll-PCR3 ®: (5' - TTA TTT TTT CCA GGT ACC CAG CAT GCC - 3'; SEQ ID NO:6).

To make the constitutive ly active Akt3 form, the coding sequence of full length Akt3 was PCR amplified by using the following primers: MyrAKT3Ha-Fl(5' - GCG CGC GAA TTC CCA CCA TGG GTA GCA ACA AGA GCA AGC CCA AGG ATG CCA GCC AGC GGC GCC GCA GCA GCG ATG TTA CCA TTG TGA AAG - 3'; SEQ ID NO: 7), which contains the Kozak sequence (CCACC), the myπstylation sequence from human src (underlined) and the first 8 ammo acids of human Akt3 (in bold), and MyrAKT3Ha-R (5' - GCG CGC GGG CCC TTA GGC GTA GTC GGG GAC GTC GTA CGG GTA TTT TTT CCA GTT ACC CAG CAT GCC - 3'; SEQ ID NO:8), which contains the coding sequence of an HA tag (in bold). The PCR product was digested with EcoR 1/Apa 1 and subcloned into the EcoR 1/Apa 1 sites of pCDNA3.1 producing pCDNA3- Myr-Akt-HA. The coding sequence of MyrAktHA was also PCR amplified and subcloned mto the Kpn 1/ EcoR 1 sites of the vector CMV6. The primers used for PCR reaction were: CMV6-AKT3cat- F (5' - CGG GGT ACC ACC ATG GGT AGC AAC AAG AGC AAG CCC AAG GAT GCC AGC CAG - 3'; SEQ ID NO:9), and CMV6-AKT3cat-R (5' - CCG GAA TTC TTA GGC GTA GTC GGG GAC GTC - 3 '; SEQ ID NO: 10). The plasmid was verified by sequencing. Example 2.2. Expression of Human AKT3

This example describes the expression of human AKT3 in tissue culture. HEK293 cells and COS-7 cells were maintained in DME media supplemented with 10% fetal bovine serum (FBS). Cells were grown in 37°C, 5%C0₂ incubator.

The plasmid CMV6-[MyrAkt3HA] was transiently transfected mto HEK293 cells. As a control, HEK293 cells were transfected with the CMV6 vector. One day prior to either transfection, cells were split to a density of 0.2x10⁶/Cm2. Transfections were carried out using LipofectAmme (Gibco BRL) according to the manufacture's instruction. Briefly, DNA was mixed in DME media (without serum or antibiotics). LipofectAmme was added (DNA:LιpofectAmme =lmg : 4ml). After brief mixing, the DNA LipofectAmme mixture was kept at room temperature for 30 minutes. Cells were washed with lxPBS, and exposed to the DNA/LipofectAmme mixture for 3 hours. After transfection, cells were washed two times with lxPBS and switched to DMEM-10%FBS media.

Twenty-four hours after transfection, cells were lysed. Lysates were lmmunoprecipitated with anti-HA antibodies, and the kmase activity of the lmmunopellets was determined using peptides denved from GSK-3, a downstream target for Aktl (Cross et al. 1995). In vitro kmase assays for Akt were earned out according to Cross et al (Cross et al, 1995) 24 hours post-transfection. Cells were washed twice m lxPBS solution, and lysed in lysis buffer (50mM Tπs/HCl, pH 7.4, ImM EDTA, ImM EGTA, 0.5mM Na₃V0₄, 0.1% β-mercaptoethanol, 1% Triton X-100, 50mM NaF, 5mM Sodium pyrophosphate, lOmM sodium glycerophosphate, 0 5mM PMSF, 2ug/ml aprotimn, 2mg/ml leupeptm, and ImM microcystin). Insoluble materials were cleared by centrifugation at 4°C for 15 minutes. Cell lysates were incubated with polyclonal anti-HA antibodies (BABCO) for 1 hour at 4°C while on a rotating platform. Protein A-Agarose beads were added to lysates for 1 hour. After immunoprecipitation, pellets were washed three times with washing solution A (lysis buffer supplemented with 0.5M NaCl), three times with washing solution B (50mM Tπs/HCl, pH7.4, 0.03% Bπj35, O.lmM EGTA and 0.1% β-mercaptoethanol), and three times with kmase buffer (20mM MOPS, pH7.2, 25mM sodium β-glycerophosphate pH7.0, ImM Na₃V0₄, ImM DTT). After washing, pellets were resuspended m 40μl kmase reaction mixture [lOOmM ATP, O.lmg/ml Crosstide substrate peptide (UBI), 20mM MgC12, lOmM protein kmase A mhibitor/PKI (UBI). and lOmCi (g-32P)-ATP] Reactions were carried out at 30°C for 30 minutes After completion of the reactions, mixtures were briefly centπfuged, and 30μl of the supernatant was loaded onto a p81 nitrocellulose paper circle (Gibco BRL). Nitrocellulose papers were washed three times with 180mM phosphonc acid (10 minutes for each washing), and two times with acetone (2 minutes for each washing). The radioactivity of the paper was monitored by Scintillation Counting Machine. Kmase activity present in CMV6[MyrAkt3HA] transfected samples was 20 times higher than that present in cells transfected with the control vector CMV6, which is similar to the background level observed for this assay (Figure IB).

To test the expression of MyrAkt3HA in transfected cells, lysates prepared from transfected cells were subjected to lmmunoblotmg with anti-HA antibodies. Cell lysates were prepared as descπbed above, and electrophoresed on SDS polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, which was then treated with blocking solution (lxPBS, 0.2% Tween 20, 5% non-fat dry milk) overnight at 4°C. Membranes were incubated with mouse monoclonal anti-HA antibodies (1 :500 dilution in blocking solution) for 3 hours at room temperature. After washing three times with blocking solution (15 minutes each), membranes were incubated with HRP-conjugated rabbit anti -mouse IgG antibodies (1:1000 dilution in blocking solution) for 1 hour at room temperature. After washing three times in blocking solution (10 minutes each) and three times in lxPBS supplemented with 0.2% Tween 20, membranes were developed in ECL (PIERCE) according to the manufacture's instruction, and exposed to Kodak X-ray film. As shown in Figure 1C, a strong -60KD band (similar to the size of MyrAktlHA, data not shown) is present in CMV6-[MyrAkt3HA] transfected samples, but not in CMV6 transfected samples (negative control). Taken together, these data demonstrate that transfection with CMV6-[MyrAkt3HA] results in functional Akt activity. Example 3: Stimulation of VEGF Expression Example 3 1. Cell culture HeLa cells (ATCC) were maintained in DME media supplemented with 10% fetal bovme serum (FBS). Cells were grown in 37°C, 5%C02 incubator. Human skeletal muscle cells (HSKMCs) and human coronary smooth muscle cells (HCSMCs) were purchased from Clonetics Corporation.

Neonatal rat cardiomyocytes were isolated using a Myocyte Isolation System (Worthmgton Biochemical Co.). Briefly, hearts collected from 1- to 3-day old rats were mmced, digested with trypsin (final concentration 50 μg/ml) overnight at 4°C, followed by digestion with collagenase at 37°C for 45 minutes. After tπturation, mixtures were filtered through cell strainer. After brief centrifugation, cells were resuspended in plating media (DMEM:M199 = 4:1, 10% heat-mactivated horse serum, 5% fetal bovme serum, lx lnsulm-transferπn-selenium supplement (Gibco BRL), and lxGentamicin, 100 μg/ml BrdU) at density of 0.3xl0⁶ cells/ml). Cells were switched to low mitogen medium (DMEM:M199 = 4: 1, lxGentamicin) 24 hours later. Example 3.2. Transfection

One day prior to transfection, cells were split at density of 0.2x10⁶ cells/cm². Transfections were carried out using lipofectAmme (Gibco BRL) according to the manufacture's instruction. Briefly, indicated DNAs were mixed in DME media (without serum or antibiotics), and lipofectAmme was added (DNA:LιpofectAmme =lμg : 4μl). After being briefly vortexed, the DNA/hpofectAmme mixture was kept at room temperature for 30 minutes. Cells were then washed with lxPBS, and exposed to the DNA/hpofectAmme mixture for 3 hours. After transfection, cells were washed two times with lxPBS and switched to DMEM-10%FBS media. 3 3 Recombinant adenovirus construction

A recombinant adenovirus containing constirutively active human Akt3 (hAkt3cak) was constructed as described Crouzet et al. (1997) (Proc. Natl. Acad. Sci. USA. Vol. 94, 1414-1419). A cDNA for constitutively active human Akt3 (comprising a myπstylation sequence from c-src ar the N-termmus) was subcloned mto pXL2996 (this plasmid is called pXL2996-hAkt3cak). The expression cassette for hAkt3cak from pXL2996-hAkt3cak was subcloned mto the shuttle vector pXL3474. This shuttle plasmid for hAkt3cak and the plasmid DNA for adenoviral-bgal (pXL3215) were introduced into bacteria JM83 cells by electroporation. After double homologous recombination, plasmid DNA for adenovιral-hAkt3cak was purified by CsCl. This DNA was lmeanzed by digestion with the restriction enzyme Pad and transfected into 293 cells using lipofectAmme. Three weeks after transfection, recombinant adenovirus containing hAkt3cak (AV-hAKT3cak) was collected, and amplified in 293 cells. Viral titer was determined using a cytoplasmic toxicity assay (CPA).

A recombinant adenovirus containing constirutively active mouse aktl (AV-mAktlcak) was prepared using standard methodology (discussed above) and provided by Dr. Kenneth Walsh (Boston, MA). Pπor to viral infection, viruses were diluted in tissue culture media at the concentration of 3xl0⁷/ml). 1ml of virus-containmg media was added to each well of a 6-well tissue culture plate, and 8ml of virus-containmg medium was added to each 100-mm culture dish. After overnight infection, excess viruses in the media were washed off with lxPBS, and cells were switched to normal media. Example 3 4 ELISA assay

Human VEGF Ehsa assays were performed using a VEGF-165 ELISA detection kit (purchased form R&D Systems, Inc., cat. DVE00). Culture media was collected and cleared by bπef centπfugation. Samples were added to each appropriate well after addition of assay diluent RD1W The plate was then allowed to incubate for two hours at room temperature. Each well was then washed with wash buffer three times. Following this, each well was treated with the provided conjugated anti-VEGF at room temperature for two hours. At this point the same aforementioned wash step was repeated. The substrate was added and incubated for twenty minutes at room temperature. Optical density was determined via a microplate reader set to 450nm, and wavelength correction was set at 540nm. Example 3.5: RNA isolation and Northern blotting.

Total RNA was isolated using Ultraspec RNA Isolation reagents (Bιotecx).Bπefly, 1ml of Ultraspec solution was added to cells in 100mm tissue culture plates. Cells were scraped from the plate and transferred mto RNase-free Eppendorf tubes. After addition of 200μl of chloroform, mixtures were vortexed and cenfrifuged at 4°C. The aqueous solution (upper layer) was collected, and RNA was precipitated with equal volume of iso-propanol. After washing with 70% ethanol and drying, RNA was dissolved in DEPC-treated water. 20μg of total RNA was separated on 1 % agarose gel. After electrophoresis and transfer, the blot was UV-cross linked.

Hybridized was performed with a p32-labeled DNA probe, generated using a Random Pπmer DNA labeling kit (Boehrmger Mannheim). After hybπdization at 65°C, the blot was washed sequentially with 2xSSC/0.1%SDS and 0.1xSSC/0.1%SDS, and exposed to Kodak X-ray film overnight. Example 3.6. Akt increases the expression of VEGF from transfected cells

HeLa cells were transfected with an expression plasmid for activated mouse Aktl (CMV6- mAktlcak), activated human Akt3 (CMV6-hAkt3) or CMV6 vector (as a control). After transfection cells were switched to low mitogen media (DMEM supplemented with 0.5% fetal bovine serum). 16 hours later, media from transfected cells was collected and subjected to ELISA for human VEGF-165. As shown in Figure 2, the VEGF level in the media of Aktl- or Akt3 -transfected cells is significantly higher than that present in the media of vector CMV6 transfected cells (as control). These data demonstrated that constirutively active Aktl or Akt3 induces VEGF-165 expression m HELA cells. Example 3 7 A V-mAktlcak and A V-hAkt3cak induce VEGF expression in human skeletal muscle and human smooth muscle cells

Human skeletal muscle cells (HSKMCs) and human coronary smooth muscle cells (HCASMCs) were infected with recombinant adneoviruses expressing either active mouse Aktl (AV- mAktlcak) or constirutively active human Akt3 (AV-hAKT3cak) As a control, cells were infected with AV-GFP, which drives the expression of green fluorescence protein One day after infection, culture media was collected, and the VEGF level in the media measured by ELISA As shown in Figure 3A, both AV-mAKTlcak and AV-hAKT3cak significantly increase VEGF-165 expression in HSKMCs, while AV-GFP infection had little or no effect Further, as shown m Figure 3B, AV- mAktl and AV-hAkt3cak induce VEGF-165 from human coronary artery smooth muscle cells (HCASMCs)

In order to evaluate the effect of Akt on VEGF messenger RNA, HCASMCs were infected with adenovirus expressing mAktlcak, hAkt3cak, or GFP As a positive control, cells were switched to hypoxic conditions for 24 hours Total RNA was isolated and subjected to Northern blot analysis for VEGF As shown in Figure 3C, hypoxia treatment dramatically induces the expression VEGF In addition, AV-mAktlcak and AV-hAkt3cak, but not AV-GFP, significantly increase the mRNA level of VEGF These data indicate that Akt increases VEGF expression by increasing the level of mRNA Example 3 8 A V-mAKTlcak and A V-hAKt3cak induces VEGF expression in cardiomyocytes

Neonatal rat cardiomyocytes were infected with adenovirus encoding mAktlcak (AV- mAktlcak), mouse wild type Aktl (AV-mAktlwt), hAkt3cak (AV-hAkt3cak) or AV-GFP (as a control) As a positive control, non-mfected cells were incubated under hypoxic conditions for 24 hours One day after infection, RNA from these cells were isolated, and VEGF expression was detected by Northern blot analysis As shown in Figure 3, AV-mAKtlcak or AV-hAkt3cak significantly increased the expression of VEGF in cardiomyocytes, while AV-GFP or AV-mAktlwt has little or no effect on VEGF-165 expression

The present invention is not to be limited in scope by the specific embodiments described herein Indeed, vaπous modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing descnption and the accompanying figures Such modifications are intended to fall withm the scope of the appended claims

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descnption

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1322-1326. Alessi DR. et al. 1996. Mechanism of activation of protein kmase B by insulin and IGF-1. EMBO J.

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1303. Chang HY et al. 1998. Activation of apoptosis signal- regulating kmase (ASKl) by the adapter protein DAXX. Science Vol281 (5384): 1860-1863. Cross DA. et al.1995. Inhibition of glycogen synthase kmase-3 by insulin mediated by protein kmase

B Nature Vol378(6559):785-789. Downward J. 1998. Mechanisms and consequences of activation of protein kmase B/Akt. Cwrr Opin

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Science Vol275(5300): 661-665. Franke TF. et al. 1995. The protein kinase encoded by the Akt proto-oncogene is a target of PDGF- activated phosphatidylmositol 3-kmase. Cell Vol81(5): 121 -lid. Franke TF. et al. 1997. PI3K: downstream AKTion blocks apoptosis. Cell Vol88(4): 435-437. Hemmings BA. 1997a. Akt signalling- linking membrane events to life and death decisions. Science

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SAPK/JNK and p38 signaling pathways. Science Vol 275: 90-94. Kauffmann-Zeh A. et al. 1997. Suppression of c-Myc-mduced apoptosis by Ras signalling through

PI(3)K and PKB. Nature Vol385(6616): 544-548. Khppel A. et al.1997. A specific product of phosphatidylmositol 3-kmase directly activates the protein kinase Akt through its pleckstrm homology domain Mol. Cell Biol Voll7(l): 338-

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USA. Vol84(14): 5034-5037. Stephens L. et al. 1998. Protein kinase B kmases that mediated phosphatidylmositol 3,4,5- tπphosphate -dependent activation of protein kinase B. Science Vol279: 710-714. Stokoe D. et al. 1997. Dual role of phosphatιdylmosιtol-3,4,5-tπphosphate in the activation of protein kinase B. Science Vol277: 567-570. Thornberry NA. et al. 1998. Caspases: enemies withm. Science Vol281 : 1312-1316.

Claims

1 A method of inducing expression of VEGF in a cell, the method compnsmg admmisteπng to the cell an Akt protein

2. The method according to claim 1, wherem the Akt protein is selected from the group consisting of Aktl, Akt2 and Akt3.

3. The method according to claim 2, wherem the Akt protein is Akt3.

4. The method according to claim 1, wherem the VEGF is selected from the group consisting of VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆, VEGF-2, VEGF-B, and VEGF-D.

5 The method according to claim 1, wherem the admmistenng comprises introducing into the cell a nucleic acid encoding the Akt protein operatively associated with an expression control sequence

6. The method according to claim 5, wherem the nucleic acid is part of a plasmid or viral vector.

7. The method according to claim 6, wherem the nucleic acid is part of a plasmid.

8. The method according to claim 6, wherem the viral vector is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes virus, and vaccinia virus.

9. The method according to claim 5, wherem the Akt is constitutively expressed withm the cell.

10. The method according to claim 1, further comprising administration of a transition metal ion and/or a vasodilator.

11. The method according to claim 5, further comprising admmistenng a nucleic acid encoding a second angiogenic factor operatively associated with an expression control sequence.

12. The method according to claim 11, wherem the second angiogenic factor is selected from the group consisting of a VEGF, acidic fibroblast growth factor, basic fibroblast growth factor, endothehal cell growth factor, and an angiopoietm.

13. The method according to claim 12, wherem the VEGF is selected from the group consisting of VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆, VEGF-2, VEGF-B, and VEGF-D.

14. The method according to claim 12, wherem the second angiogenic factor is endothehal cell growth factor.

15. The method according to claim 2, wherem at least two forms of Akt protein are administered to the cell

16. The method according to claim 1 , wherem the cell is in a patient suffering from an ischemic condition.

17. The method according to claim 16, wherem the ischemic condition is cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, myocardial ischemia, or ischemic, idiopathic or hypertrophic cardiomyopathy.

18 A method of inducing expression of VEGF in cells of a patient suffering from an ischemic condition, the method comprising administering to the patient an Akt protein.

19. The method according to claim 18, wherein the Akt protein is selected from the group consisting of Aktl, Akt2 and Akt3

20. The method according to claim 19, wherem the Akt protein is Akt3

21. The method according to claim 18, wherein the VEGF is selected from the group consisting of VEGF₁₂₁, VEGF₁₆-, VEGF₁₈₉, VEGF₂₀₆, VEGF-2, VEGF-B, and VEGF-D.

22. The method according to claim 18, wherem a nucleic acid encoding the Akt protein operatively associated with an expression control sequence is administered to the patient

23. The method according to claim 22, wherem the nucleic acid is part of a plasmid or viral vector.

24. The method according to claim 23, wherem the nucleic acid is part of a plasmid.

25. The method according to claim 23, wherem the viral vector is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes virus, and vaccinia virus

26. The method according to claim 22, wherem the Akt is constitutively expressed withm the cell.

27. The method according to claim 18, further comprising administration of a transition metal ion and/or a vasodilator to the patient.

28. The method according to claim 18, wherein the ischemic condition is cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, myocardial ischemia, or ischemic, idiopathic or hypertrophic cardiomyopathy.

29. The method according to claim 22, further compnsmg admmistenng a nucleic acid encoding a second angiogenic factor operatively associated with an expression control sequence.

30 The method according to claim 29, wherem the second angiogenic factor is selected from the group consisting of a VEGF, acidic fibroblast growth factor, basic fibroblast growth factor, endothehal cell growth factor, and an angiopoietm.

31. The method according to claim 30, wherein the VEGF is selected from the group consisting of VEGF,₂i, VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆, VEGF-2, VEGF-B, and VEGF-D.

32. The method according to claim 30, wherem the second angiogenic factor is endothehal cell growth factor.

33. The method according to claim 16, wherem at least two forms of Akt protein are administered to the patient.

34. A pharmaceutical composition compnsmg a nucleic acid encoding an Akt protein, a transition metal and or a vasodilator, and a pharmaceutically acceptable vehicle.

35. The composition according to claim 34, where the nucleic acid is part of a plasmid or viral vector.

36. The composition according to claim 35, wherem the nucleic acid is part of a plasmid.

37. The composition according to claim 55, wherem the viral vector is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes virus, and vaccinia virus.

38. A method of inhibiting angiogenesis in a patient suffering from a tumor, the method compnsmg inhibiting the level of Akt activity in the patient, thereby inhibiting production of VEGF.

39. The method according to claim 38, comprising introducing an Akt antisense nucleic acid mto cells of the patient under conditions wherein the antisense nucleic acid hybπdizes under intracellular conditions to an Akt mRNA.

40. The method according to claim 38, comprising introducing an intracellular binding protein that specifically binds Akt into a patient's cells at a level sufficient to bind to and inactivate Akt.

41. The method according to claim 40, wherein the intracellular binding protein is a single chain Fv antibody (scFv).

42. The method according to claim 38, comprising introducing a nucleic acid encoding a dominant negative form of an Akt.