WO2003086447A1 - Methods for modulating neovascularization - Google Patents

Abstract

A method of promoting or inhibiting angiogenesis in an animal, including human and non-human mammals, by administering to the animal SCM113 protein, or a variant, fragment, or derivative thereof.

Description

METHODS FOR MODULATING NEOVASCULARIZATION

This application claims the benefit of U.S. Provisional Application No. 60/371,209, filed April 9, 2002, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

This invention relates to modulating neovascularization in a mammal. More particularly, this invention relates to promoting or inhibiting angiogenesis or vasculogenesis in a mammal by administering to the mammal SCM113 protein, or a fragment, derivative, or variant thereof. BACKGROUND OF THE INVENTION

The formation of new blood vessels in the adult is commonly believed to occur by sprouting from an existing vessel and is termed angiogenesis. This process requires local destabilization of the vessel with subsequent endothelial cell migration, proliferation, remodeling, and formation of tight attachments with the extracellular matrix and supporting mural cells. New capillary invasion into a previously avascular zones is stimulated by ischemic or hypoxic conditions. The lack of oxygen under these conditions leads to the upregulation of the transcription factor, hypoxia inducible factor-1 (HIF-1). HIF-1 is the primary regulator of oxygen homeostasis in mammalian cells and among its various activities, promotes the production of the key angiogenic molecule, VEGF. VEGF, acting via its cognate receptors, initiates a cascade of events that leads to the formation of a new capillary network.

A distinct mechanism of blood vessel formation known to occur during early embryonic development is termed vasculogenesis. This early morphogenic process occurs via the differentiation of angioblasts (endothelial cell precursors) into blood islands that fuse to form the primitive vascular network. There is now increasing evidence to suggest that angioblast-like endothelial cell precursors derived from the bone marrow may circulate in the adult vasculature and contribute to physiological and pathological neovascularization in the adult. This concept provides the possibility of intriguing alternative therapeutic options as well as the potential to discover new targets for the effective modulation of neovascularization.

Neovascularization is an essential part of normal development but in the adult it normally occurs only as part of the female estrus cycle, during pregnancy, and as part of tissue repair and remodeling. Pro-angiogenesis therapy seeks to enhance the natural process of tissue repair and remodeling with primary emphasis on ischemic vascular diseases. Pathological neovascularization has been implicated in a number of diseases including age- related macular degeneration, diabetic retinopathy, and rheumatoid arthritis. However, the major focus of anti-angiogenesis therapies today is on cancer. Continued tumor growth requires a concomitant increase in blood supply to nourish the cancer cells, and the ability of these cells to switch to a pro-angiogenic phenotype is an early event in the progression of most cancers. The tumor vasculature is an attractive and novel therapeutic target since endothelial cells should not be capable of developing resistance to anti-angiogenic agents. Furthermore, there is evidence to suggest that the tumor vasculature is sufficiently different from normal vessels that strategies can be devised to target tumors specifically.

Several studies have shown recently that endothelial progenitor cells, or EPCs, are present in the population of hematopoietic progenitor cells, and that these cells may contribute to therapeutic angiogenesis. Thus, EPCs may contribute to the angiogenic process and serve as a useful model for the discovery of proteins or polypeptides involved in angiogenesis and of genes encoding such proteins or polypeptides.

SUMMARY OF THE INVENTION The present disclosure supports for the first time the surprising discovery that the hemopoietic stem cell (HSC) regulatory gene product, SCMl 13 (PCT/EP99/05566 (WO 00/08145)), increases migration of human umbilical vein endothelial cells (HUVEC) and that SCMl 13 overexpression results in enhanced endothelial cell proliferation in response to angiogenic growth factors. Thus, the present invention is directed to the use of SCMl 13 protein, or an analogue, fragment, or derivative thereof, to promote or inhibit neovascularization in an animal.

According, the present invention provides a method for promoting neovascularization in an animal comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to promote neovascularization in said animal.

The present invention also provides a method for preventing or treating congestive heart failure in an animal, comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to prevent or treat congestive heart failure in said animal.

The present invention additionally provides a method for preventing or treating myocardial ischemia in an animal comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to prevent or treat myocardial ischemia in said animal.

The present invention further provides a method for treating ischemia-reperfusion injury in an animal comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to treat ischemia-reperfusion injury in said animal. The present invention still further provides a method for treating a peripheral arterial disease in an animal comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to treat the peripheral arterial disease in said animal. In one embodiment of a method of the invention, said SCMl 13 protein, or variant, fragment, or derivative thereof comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2.

In another embodiment of a method of the invention, said SCMl 13 protein, or variant, fragment, or derivative thereof comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2.

In yet another embodiment of a method of the invention, said SCMl 13 protein, or variant, fragment, or derivative thereof comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:2.

Still another embodiment of a method of the invention comprises administering to said animal SCMl 13 protein comprising an amino acid sequence as shown in SEQ ID NO:2 or an active fragment thereof.

In one embodiment of a method of the invention, said SCMl 13 protein, or variant, fragment, or derivative thereof is administered to said animal as a recombinant protein. In exemplary embodiments, said protein is administered orally, intravenously, intramuscularly, intraperitoneally, or intrathecally.

In another embodiment of a method of the invention, said SCMl 13 protein, or variant, fragment, or derivative thereof is administered by administering a vector comprising a nucleotide sequence encoding said SCMl 13 protein, or variant, fragment, or derivative thereof. In exemplary embodiments, said vector is a plasmid, a lipid formulation, or a viral vector. Preferably, said vector is administered orally, intravenously, intramuscularly, intraperitoneally, or intrathecally.

In a preferred embodiment of a method of the invention, said vector is a viral vector, hi exemplary embodiments, said viral vector is an adeno associated viral vector, a plasmid, or an adenoviral vector. In another exemplary embodiment, said viral vector is a retroviral vector, such as an MoMLV-vector. In another exemplary embodiment, said viral vector is a lentiviral vector, such as a lentiviral vector derived from a lentivirus selected from the group consisting of HIN, BIV, EIAV, FIV, and SIV. In one embodiment of a method of the invention, said nucleotide sequence encoding said SCMl 13 protein, or variant, fragment, or derivative thereof is at least 85% identical to SEQ ID NO: 1.

In another embodiment of a method of the invention, said nucleotide sequence encoding said SCMl 13 protein, or variant, fragment, or derivative thereof is at least 90% identical to SEQ ID NOrl.

In yet another embodiment of a method of the invention, said nucleotide sequence encoding said SCMl 13 protein, or variant, fragment, or derivative thereof is at least 95% identical to SEQ ID NO:l.

In still another embodiment of a method of the invention, said nucleotide sequence comprises SEQ ID NO:l .

According to another embodiment of a method of the invention, said vector further comprises a nucleotide sequence encoding a protein selected from the group consisting of VEGF, FGF, IGF, an angiopoietin, PD-EGF, TGF-/3, HIFl-o; nitric oxide synthase, MCP-1, Interleukin-8, ephrins, NAP-2, ENA-78, GROW- ct, and an active fragment of tyrosyl-tRNA synthase.

According to one embodiment of a method of the invention, an adenoviral vector is administered to said animal in an amount of from about 5x10⁹ plaque forming units to about

5x10 particles per kilogram.

According to another embodiment of a method of the invention, an adenoviral vector is administered to said animal in an amount of from about 5xl0¹⁰ plaque forming units to about lxl 0¹³ plaque forming units.

According to another embodiment of a method of the invention, an adenoviral vector is administered to said animal in an amount of from about lxl 0¹¹ transducing units to about 10¹³ transducing units. According to one embodiment of a method of the invention, a lentiviral vector is administered to said animal in an amount of from about lxlO⁴ transducing units to about 10^π transducing units. According to another embodiment of a method of the invention, a lentiviral vector is administered to said animal in an amount of from about lxlO⁵ transducing units to about lxlO⁸ transducing units.

According to still another embodiment of a method of the invention, a lentiviral vector is administered to said animal in an amount of from about lxl 0⁵ transducing units to about lxl 0⁶ transducing units.

According to one method of the invention, said animal is a mammal. Preferably, said mammal is a primate. More preferably, said primate is a human.

In another embodiment of a method of the invention, said SCMl 13 protein, or variant, fragment, or derivative is administered to said animal by transducing cells of blood vessels with an expression vehicle comprising a gene construct encoding said SCMl 13 protein, or analogue, fragment or derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic of the retroviral vector SCMl 13 and the control vector MIE. Figure 2 is a graph of human umbilical vein endothelial cell (HUVEC) migration in response to FGF. A tilt assay was done in a 12 well plate with HUVEC cells. Data are expressed as mean + SD from triplicate samples.

Figure 3 A and 3B are graphs of HUVEC cell migration in tilt assays showing migration of HUVEC cells transduced with SCMl 13 or MIE in response to 20 ng/ml hVEGF (Figure 3 A) or 2ng/ml FGF (Figure 3B).

Figure 4 shows graphs of HUVEC cell migration in a transwell assay of HUVEC cells transduced with SCMl 13 in response to lOng/mg of FGF.

DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING The Sequence Listing associated with the instant disclosure is hereby incorporated by reference into the instant disclosure. The following is a description of the sequences contained in the Sequence Listing:

SEQ ID NO:l is a cDNA sequence of SCMl 13.

SEQ ID NO:2 is the amino acid sequence of the native human SCMl 13 protein. SEQ ID NO:3 is a PCR primer. DEFINITIONS

"SCMl 13" means the hemopoietic stem cell (HSC) regulatory gene product shown in Figure 6 of PCT/EP99/05566 (WO 00/08145, incorporated herein by reference. The amino acid sequence of the native unmodified human SCMl 13 protein is shown in the sequence listing as SEQ ID NO:2, and a nucleotide sequence encoding it is shown as SEQ ID NO:l.

"Neovascularization" means the proliferation of blood vessels in tissue and includes angiogenesis and vasculogenesis. The term "angiogenesis," as used herein, means new blood vessel formation that includes local endothelial cell migration, proliferation, and remodeling as well as the investment of circulating precursor cells to generate new blood vessels. The term "vasculogenesis" means blood vessel formation known to occur during early embryonic development that occurs via the differentiation of angioblasts (endothelial cell precursors) into blood islands that fuse to form the primitive vascular network. "Cell culture" encompasses both the culture medium and the cultured cells.

The phrase "isolating a polypeptide from the cell culture" encompasses isolating a soluble or secreted polypeptide from the culture medium as well as isolating an integral membrane protein from the cultured cells.

"Cell extract" includes culture media, especially spent culture media from which the cells have been removed. A cell extract that contains the DNA or protein of interest should be understood to mean a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest.

"Plasmid" is an autonomous, self-replicating extrachromosomal DNA molecule and is designated by a lower case "p" preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 °C. are ordinarily used, but can vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment. The nucleotides present in various DNA and RNA fragments are designated herein by the standard single letter designations (A, T, C, G, U) used in the art.

"Polynucleotide" embodying the present invention can be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA can be double-stranded or single-stranded, and if single stranded can be the coding strand or non- coding (anti-sense) strand.

The term "Polynucleotide encoding a polypeptide" encompasses a polynucleotide that includes only coding sequence for the polypeptide as well as a polynucleotide that includes additional coding and/or non-coding sequence.

"Oligonucleotides" refers to either a single stranded polynucleotide or two complementary polynucleotide strands that can be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an A TP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Amino acid residue" refers to an amino acid that is part of a polypeptide. The amino acid residues described herein are preferably in the L" isomeric form. However, residues in the D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. All amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. A dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino- terminal group such as NH₂ or to a carboxyl-terminal group such as COOH.

In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1 87, The

Benjamin/Cummings Pub. Co., p.224). Such substitutions are preferably made in accordance with those set forth as follows: Original residue Conservative substitution(s)

Ala Gly; Ser

Arg Lys

Asn Gin; His Cys Ser

Gin Asn

Glu Asp

Gly Ala; Pro

His Asn; Gin lie Leu; Val

Leu He; Val

Lys Arg; Gin; Glu

Met Leu; Tyr; He

Phe Met; Leu; Tyr Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val He; Leu Other substitutions are also permissible and can be determined empirically or in accord with known conservative substitutions.

As used herein, a polypeptide "variant" or "derivative" refers to a polypeptide that is a mutagenized form of a polypeptide or one produced through recombination but that still retains a desired activity, such as the ability to bind to a ligand or a nucleic acid molecule or to modulate transcription. In particular, a derivative of SCMl 13 refers to an alteration of the native SCMl 13 to one produced through amino acid substitution, addition, or deletion that retains SCMl 13 's ability to increase migration of human umbilical vein endothelial cells (HUVEC) and results in enhanced endothelial cell proliferation in response to angiogenic growth factors. Thus, a derivative of SCMl 13 includes a polypeptide in which one or more wild-type amino acids are substituted with alternate amino acids, and includes primary sequence changes. For example, a derivative of SCMl 13 includes a polypeptide having at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and yet more preferably at least 99% identity with SEQ HD NO:2. A derivative of SCMl 13 also includes a polypeptide encoded by a nucleotide sequence having at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and yet more preferably at least 99% identity with SEQ ID NO:l. A derivative of SCM113 also includes a polypeptide encoded by a nucleotide sequence having a complement that hybridizes under stringent conditions to SEQ ID NO:l. A derivative of SCMl 13 also includes truncations of SCMl 13 that retain SCMl 13's ability to increase migration of human umbilical vein endothelial cells (HUVEC) and result in enhanced endothelial cell proliferation in response to angiogenic growth factors. Such truncations may include N-terminal deletions, C-terminal deletions, and internal deletions of amino acids. In an alternate embodiment, a variant or derivative of SCMl 13 may exhibit an antiangiogenic function acting as a dominant-negative protein.

As used herein, the term "nucleic acid cassette" refers to the genetic material of interest which can express a protein, or a peptide, or RNA after it is incorporated transiently, permanently or episomally into a cell. The nucleic acid cassette is positionally and sequentially oriented in a vector with other necessary elements such that the nucleic acid in the cassette can be transcribed and, when necessary, translated in the cell.

"Complementing plasmid" describes plasmid vectors that deliver nucleic acids into a packaging cell line for stable integration into a chromosome in the cellular genome. "Delivery plasmid" is a plasmid vector that carries or delivers nucleic acids encoding a therapeutic gene or gene that encodes a therapeutic product or a precursor thereof or a regulatory gene or other factor that results in a therapeutic effect when delivered in vivo in or into a cell line, such as, but not limited to a packaging cell line, to propagate therapeutic viral vectors. A variety of vectors are described herein. For example, one vector is used to deliver particular nucleic acid molecules into a packaging cell line for stable integration into a chromosome. These types of vectors are generally identified herein as complementing plasmids. A further type of vector described herein carries or delivers nucleic acid molecules in or into a cell line (e.g., a packaging cell line) for the purpose of propagating therapeutic viral vectors; hence, these vectors are generally referred to herein as delivery plasmids. A third "type" of vector described herein is used to carry nucleic acid molecules encoding therapeutic proteins or polypeptides or regulatory proteins or are regulatory sequences to specific cells or cell types in a subject in need of treatment; these vectors are generally identified herein as therapeutic viral vectors or recombinant adenoviral vectors or viral Ad-derived vectors and are in the form of a virus particle encapsulating a viral nucleic acid containing an expression cassette for expressing the therapeutic gene.

The terms "homology" and "identity" are often used interchangeably. In this regard, degree of homology or identity can be determined, for example, by comparing sequence information using a GAP computer program. The GAP program utilizes the alignment method of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman, Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" can be determined using known computer algorithms such as the "FAST A" program, using for example, the default parameters as in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988). Alternatively the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. In general, sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, (1988); Smith, D.W., ed., Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Griffin, A.M., and Griffin, H.G., eds., Computer Analysis of Sequence Data, Parti, HumanaPress, New Jersey, (1994); vonHeinje, G., Sequence Analysis in Molecular Biology, Academic Press, (1987); and Gribskov, M. and Devereux, J., eds., Sequence Analysis Primer, M Stockton Press, New York, (1991)). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H. & Lipton, D., SIAMJ. Applied Math.

48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Martin J. Bishop, ed., Guide to Huge Computers, Academic Press, San Diego, (1994), and Carillo, H. & Lipton, D., SIAMJ. Applied~ath. 48:1073 (1988). Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al, Nucleic Acids Research 12(1):387 (1984), BLASTP, BLASTN, FASTA (Atschul, S.F., et al, J. Malec. Biol 215:403 (1990)).

The term "identity" represents a comparison between a test and a reference polypeptide or polynucleotide. For example, a test polypeptide can be defined as any polypeptide that is 90% or more identical to a reference polypeptide. As used herein, the term at least "90% identical to" refers to percent identities from 90 to 99.99 relative to the reference polypeptides. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they' can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, or deletions.

The phrase "hybridizing to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

"Stringent hybridization conditions" and "stringent wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part 1 chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5°C to 20°C (preferably 5°C) lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Typically, under highly stringent conditions a probe will hybridize to its target subsequence, but to no other sequences.

The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T_m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2xSSC wash at 65°C for 15 mmutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lxSSC at 45°C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6xSSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

The terms "gene therapy" and "genetic therapy" refer to the transfer of heterologous DNA to the certain cells, target cells, of a mammal, particularly a human, with a disorder or conditions for which such therapy is sought. The DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced. Alternatively, the heterologous DNA can in some manner mediate expression of DNA that encodes the therapeutic product, it can encode a product, such as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product. Genetic therapy can also be used to nucleic acid encoding a gene product replace a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced. The introduced nucleic acid can encode a therapeutic compound, such as a growth factor inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefor, that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time. The heterologous DNA encoding the therapeutic product can be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.

"Heterologous DNA" is DNA that encodes RNA and proteins that are not normally produced in vivo by the cell in which it is expressed or that mediates or encodes mediators that alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes. Heterologous DNA can also be referred to as foreign DNA. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which is expressed is herein encompassed by heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies. Antibodies that are encoded- by heterologous DNA can be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced. Hence, "heterologous DNA" or "foreign DNA", refers to a DNA molecule not present in the exact orientation and position as the counterpart DNA molecule found in the corresponding wild-type adenovirus. It can also refer to a DNA molecule from another organism or species (i.e., exogenous) or from another Ad serotype.

"Therapeutically effective DNA product" is a product that is encoded by heterologous DNA so that, upon introduction of the DNA into a host, a product is expressed that effectively ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease or that cures said disease. Typically, DNA encoding the desired heterologous DNA is cloned into a plasmid vector and introduced by routine methods, such as calcium-phosphate mediated DNA uptake or microinjection, into producer cells, such as packaging cells. After amplification in producer cells, the vectors that contain the heterologous DNA are introduced into selected target cells.

"Expression or delivery vector" refers to any plasmid or virus into which a foreign or heterologous DNA can be inserted for expression in a suitable host cell- i.e., the protein or polypeptide encoded by the DNA is synthesized in the host cell's system. Vectors capable of directing the expression of DNA segments (genes) encoding one or more proteins are referred to herein as "expression vectors." Also included are vectors that allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase. "Gene" is a nucleic acid molecule whose nucleotide sequence encodes RNA or polypeptide. A gene can be either RNA or DNA. Genes can include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). "Isolated" with reference to a nucleic acid molecule, polypeptide, or other biomolecule, means that the nucleic acid or polypeptide has separated from the genetic environment from which the polypeptide or nucleic acid was obtained. It can also mean altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated. Also intended as an "isolated polypeptide" or an "isolated polynucleotide" are polypeptides or polynucleotides that have been purified, partially or substantially, from a recombinant host cell or from a native source. For example, a recombinantly produced version of a compounds can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). The terms "isolated" and "purified" are sometimes used interchangeably. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. By "isolated polynucleotide" is meant that the nucleic acid is free of the coding sequences of those genes that, in the naturally occurring genome of the organism (if any) immediately flank the gene encoding the nucleic acid of interest. Isolated DNA can be single- stranded or double-stranded, and can be genomic DNA, cDNA, recombinant hybrid DNA, or synthetic DNA. It can be identical to a native DNA sequence, or can differ from such sequence by the deletion, addition, or substitution of one or more nucleotides.

"Isolated" or "purified" as it refers to preparations made from biological cells or hosts means any cell extract containing the indicated DNA or protein including a crude extract of the DNA or protein of interest. For example, in the case of a protein, a purified preparation can be obtained following an individual technique or a series of preparative or biochemical techniques and the DNA or protein of interest can be present at various degrees of purity in these preparations. The procedures can include for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange change chromatography, affinity chromatography, density gradient centrifugation and electrophoresis. A preparation of DNA or protein that is "substantially pure" or "isolated" means a preparation free from naturally occurring materials with which such DNA or protein is normally associated in nature. "Essentially pure" should be understood to mean a "highly" purified preparation that contains at least 95 % of the DNA or protein of interest. "Packaging cell line" is a cell line that provides a missing gene product or its equivalent.

"Adenovirus viral particle" is the minimal structural or functional unit of a virus. A virus can refer to a single particle, a stock of particles or a viral genome. The adenovirus (Ad) particle is relatively complex and can be resolved into various substructures.

"Post-transcription regulatory element (PRE)" is a regulatory element found in viral or cellular messenger RNA that is not spliced, i.e. intronless messages. Examples include, but are not limited to, human hepatitis virus, woodchuck hepatitis virus, the TK gene and mouse histone gene. The PRE can be placed before a polyA sequence and after a heterologous DNA sequence

"Pseudotyping" describes the production of adenoviral vectors having modified capsid protein or capsid proteins from a different serotype than the serotype of the vector itself. One example is the production of an adenovirus 5 vector particle containing an Ad37 fiber protein. This can be accomplished by producing the adenoviral vector in packaging cell lines expressing different fiber proteins.

"Promoters of interest herein" can be inducible or constitutive. Inducible promoters will initiate transcription only in the presence of an additional molecule; constitutive promoters do not require the presence of any additional molecule to regulate gene expression, a regulatable or inducible promoter can also be described as a promoter where the rate or extent of RNA polymerase binding and initiation is modulated by external stimuli. Such stimuli include, but are not limited to various compounds or compositions, light, heat, stress and chemical energy sources. Inducible, suppressible and repressible promoters are considered regulatable promoters. Preferred promoters herein, are promoters that are selectively expressed in ocular cells, particularly photoreceptor cells.

"Receptor" refers to a biologically active molecule that specifically binds to (or with) other molecules. The term "receptor protein" can be used to more specifically indicate the proteinaceous nature of a specific receptor.

"Recombinant" refers to any progeny formed as the result of genetic engineering. This can also be used to describe a virus formed by recombination of plasmids in a packaging cell. "Transgene" or "therapeutic nucleic acid molecule" includes DNA and RNA molecules encoding an RNA or polypeptide. Such molecules can be "native" or naturally derived sequences; they call also be "non-native" or "foreign" that are naturally or recombinantly derived. The term "transgene," which can be used interchangeably herein with the term "therapeutic nucleic acid molecule," is often used to describe a heterologous or foreign

(exogenous) gene that is carried by a viral vector and transduced into a host cell. Therapeutic nucleotide nucleic acid molecules include antisense sequences or nucleotide sequences that can be transcribed into antisense sequences. Therapeutic nucleotide sequences (or transgenes) all include nucleic acid molecules that function to produce a desired effect in the cell or cell nucleus into which said therapeutic sequences are delivered. For example, a therapeutic nucleic acid molecule can include a sequence of nucleotides that encodes a functional protein intended for delivery into a cell which is unable to produce that functional protein.

"Promoter region" refers to the portion of DNA of a gene that controls transcription of the DNA to which it is operatively linked. The promoter region includes specific sequences of DNA that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of the RNA polymerase. These sequences can be cis acting or can be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, can be constitutive or regulated.

"Operatively linked" means that the sequences or segments have been covalently joined into one piece of DNA, whether in single or double stranded form, whereby control sequences on one segment control expression or replication or other such control of other segments. The two segments are not necessarily contiguous, however. "Complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.

"Label" and" Indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems. DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there is provided a method of promoting angiogenesis in an animal. The method comprises administering to the animal SCMl 13 protein, or a variant, fragment, or derivative thereof. The SCMl 13 protein, or variant, fragment, or derivative thereof is administered in an amount effective to promote or inhibit angiogenesis in the animal. The SCMl 13 protein, or variant, fragment, or derivative thereof, or polynucleotide encoding SCMl 13 protein or a variant, fragment, or derivative thereof, may be administered in combination with other angiogenic proteins or polynucleotides encoding other angiogenic proteins such as, but not limited to, VEGF, FGF, IGF, angiopoietins, PD-EGF, TGFβ, HTFl-α, nitric oxide synthase, MCP-1, Interleukin-8, ephrins, NAP-2, ENA-78, GROW-2, and fragments of tyrosyl-tRNA synthetase that have angiogenic activity as disclosed inPCT/USOl/08966 (WO 01/74841) and PCT/USO 1/08975 (WO 01/75078).

The findings that SCMl 13 increases migration of human umbilical vein endothelial cells (HUVEC) and that SCMl 13 overexpression results in enhanced endothelial cell proliferation in response to angiogenic growth factors indicate that SCMl 13 can be used to promote or inhibit neovascularization in an animal. The SCMl 13 or derivatives can be delivered as a recombinant protein or by gene therapy vectors including but not limited to adenoviral, AAV, HSV vector, retroviral, lentiviral, and plasmid vectors. The invention further includes small molecules or other drugs based on this interaction.

Accordingly, one aspect of the present invention is directed to the use of SCMl 13, its derivatives, modifications, or small molecules or drugs based on this interaction for the modulation of neovascularization.

The SCMl 13 protein, or variant, fragment, or derivative thereof may be prepared by techniques known to the skilled in the art. For example, the SCMl 13 protein or variant, fragment, or derivative thereof may be prepared by an automated peptide or protein synthesizer. Alternatively, the SCMl 13 protein or variant, fragment, or derivative thereof may be prepared by genetic engineering techniques.

In one embodiment, the SCMl 13 protein or variant, or fragment, or derivative thereof is administered to the animal, or to cells of blood vessels, by delivery of a polynucleotide comprising a gene construct encoding the SCMl 13 protein or variant, or fragment, or derivative thereof. Preferably, the polynucleotide comprises an appropriate expression vehicle.

In one embodiment, the SCMl 13 protein or variant, or fragment, or derivative thereof is a(--ministered to a cell in vitro. Preferably the cell is mammalian and most preferably human. The invention further provides gene transfer vectors encoding SCMl 13 protein or variant, or fragment, or derivative thereof and methods of gene transfer and expression which can be used to alter the expression patterns of genes in the study of gene function in particular cell types.

Viral Delivery systems Viral transduction methods for delivering nucleic acid constructs to cells are contemplated herein. Suitable DNA viral vectors for use herein includes, but are not limited to an adenovirus (Ad), adeno-associated virus (AAV), herpes virus, vaccinia virus or a polio virus. A suitable RNA virus for use herein includes but is not limited to a retro virus or Sindbis virus. It is to be understood by those skilled in the art that several such DNA and RNA viruses exist that may be suitable for use herein. Adenoviral vectors have proven especially useful for gene transfer into eukaryotic cells and are widely available to one skilled in the art and is suitable for use herein.

Adeno-associated virus (AAV) has been used as a gene transfer system with applications in gene therapy. See U.S. Patents Nos. 5,139,941; 5,436,146; and 5,622,856. Herpes simplex virus type-1 (HSV-1) vectors are available and are especially useful in the nervous system because of its neurotropic property. See U.S. Patent No. 5,288,641. Vaccinia viruses, of the poxvirus family, have also been developed as expression vectors. Each of the above-described vectors is widely available and is suitable for use herein.

Retroviral vectors are capable of infecting a large percentage of the target cells and integrating into the cell genome. Preferred retroviruses include lentiviruses, and also include, but are not limited to, HIV, BIV and SIV. See U.S. Patents Nos. 5,665,577; 5,994,136; 6,013,516; 5,672,510; 5,707,865 and 5,817,491, as well WO 01/44458 and PCT/US03/03307.

Various viral vectors that can be used for gene therapy as taught herein include adenovirus (See U.S. Patent No.5,935,935), herpes virus, vaccinia, adeno-associated virus (AAV), or, preferably, an RNA virus such as a retro virus, and also include a modified viral vector, such as an adenovirus, known as a "gutless" vector. Preferably, the retroviral vector is a derivative of a murine or avian retro virus, or is a lentiviral vector. The preferred retroviral vector is a lentiviral vector. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSN), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a zinc finger derived- DΝA binding polypeptide sequence of interest into the viral vector, along with another gene that encodes the ligand for a receptor on a specific target cell, for example, the vector is made target specific.

Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a protein. Preferred targeting is accompUshed by using an antibody to target the retroviral vector. Those of skill in the art know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the zinc finger-nucleotide binding protein polynucleotide. Because recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retro virus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RΝA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include but are not limited to ^"92, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as ΝIH 3T3 cells, to produce large quantities of chimeric retroviral virions.

Additionally, preferred vectors include adenoviral vectors (See, Frey, B.M. et al., Blood, 91:2781 (1998); Kochanek, Hum. Gen. Ther., 10:2459-2461 (1999); Sandig, et al., PNAS, 97(3):1002-1007 (2000); Reddy, et al., Mol. Ther., 5(l):63-73 (2002); and WO95/27071 and WO96/18418) and adeno-associated viral vectors (See, Chatterjee et al., Current Topics in

Microbiol. and Immunol., 218:61-73, 1996). Also, reference is made to Shenk, Chapter 6, 161- 178, Breakfield, et al., Chapter 8 201-235; Kroner-Lux et al., Chapter 9 235-256 in Stem Cell Biology and Gene Therapy, eds. Quesenberry et al., John Wiley & Sons, 1998 and U.S. Pat. Nos. 5,693,531, and 5,691,176. The use of adenovirus-derived vectors may be advantageous in certain situations because they are not capable of infecting non-dividing cells, and unlike retroviral DNA, the adenoviral DNA is not integrated into the genome of the target cell. Further, the capacity to carry foreign DNA is much larger in adenoviral vectors than retroviral vectors. The adeno associated viral vectors are another useful delivery system. The DNA of this virus may be integrated into non-dividing cells, and a number of polynucleotides have been successfully introduced into different cell types using adeno-associated viral vectors.

The adenoviral vector which is employed may, in one embodiment, be an adenoviral vector which includes essentially the complete adenoviral genome (Shenk et al., Curr. Top. Microbiol. Immunol., 111(3): 1-39 (1984). Alternatively, the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted. In one embodiment, the adenoviral vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence encoding a SCMl 13 protein, or an analogue, fragment, or derivative thereof, and a promoter controlling the DNA sequence encoding a SCMl 13 protein, or an analogous, fragment, or derivative thereof. The vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter. In one embodiment, the vector also is free of at least a portion of at least one DNA sequence selected from the group consisting of the E2 and E4 DNA sequences. In another embodiment, the vector is free of at least the majority of the adenoviral El and E3 DNA sequences, and is free of a portion of the other of the E2 and E4 DNA sequences. In still another embodiment, the gene in the E2a region that encodes the 72 kilodalton binding protein is mutated to produce a temperature sensitive protein that is active at 32°C, the temperature at which the viral particles are produced. This temperature sensitive mutant is described in Ensinger, et al., J. Virology, 10:328-339 (1972), Van der Vliet et al, J. Virology, 15:348-354 (1975), and Friefeld, et al, Virology, 124:380-389 (1983). In another embodiment, the adenoviral vector is free of all or a portion of each of the adenoviral El and E4 DNA sequences, or is free of all or a portion of each of the adenoviral El and E2 DNA sequences, or is free of all or a portion of each of the El, E2, and E4 DNA sequences. Such vectors, wherein the vector is free of all or a portion of each of the adenoviral El and E4 DNA sequences, or is free of all or a portion of each of the adenoviral El and E2 DNA sequences, or is free of all or a portion of the El, E2 and E4 DNA sequences, and the complementing cell lines, also are described in PCT Application No. WO96/18418, published June 20, 1996, the contents of which are incorporated herein by reference. In another embodiment, the adenoviral vector is free of all adenoviral coding regions. This "gutless" adenoviral vector includes an adenoviral 5' ITR, an adenoviral packaging signal, a DNA sequence encoding SCMl 13 or an analogue, fragment, or derivative thereof, and an adenoviral 3' ITR (Sandig, et al, PNAS, 97(3): 1002-1007 (2000); Reddy, et al., Mol Ther., 5(l):63-73 (2002)): The vector contains from about 26 kb to about 38 kb, preferably 28 kb to 32 kb, and may include one or more genomic elements. In another embodiment the vector is an oncolytic adenoviral vector (US Patent 5,998,205 and US Patent 5,677,178). Alternatively, the adenoviral vector may have a modified fiber protein whereby the adenoviral vector is "targeted" to a specific cell. Representative examples of such adenoviral vectors are disclosed in U.S. Patent No. 5,543,328. The various adenoviral vectors may include promoters other than a SCMl 13 promoter, such as tissue-specific promoters. The vector also may include, in addition to a DNA sequence encoding a SCMl 13 protein, or an analogue, fragment, or derivative thereof, DNA sequences encoding additional proteins which facilitate the generation of new blood vessels, such as, but not limited to, vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), IGFs, angiopoietins, including angiopoietin 1, and angiopoietin 2, TGF-β, hypoxia inducible factors (HIFs) such as HIFl-α, monocyte chemoattractant proteins (MCPs) such as MCP-1, nitric oxide synthase, ephrins, such as ephrin B2, and other angiogenic genes, platelet derived endothelial growth factor, and Interleukin-8. Nonviral Delivery systems

"Non-viral" delivery techniques for gene therapy include DNA-ligand complexes, adeno virus-ligand-DNA complexes, direct injection of DNA, CaPO precipitation, gene gun techniques, electroporation, liposomes and lipofection. Any of these methods are available to one skilled in the art and would be suitable for use herein. Other suitable methods are available to one skilled in the art, and it is to be understood that the herein may be accomplished using any of the available methods of transfection.

Another targeted delivery system is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, which are preferred. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 jLtm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al, Trends Biochem. Sci., 6:77, 1981).

Lipofection may be accomplished by encapsulating an isolated nucleic acid molecule within a liposomal particle and contacting the liposomal particle with the cell membrane of the target cell. Liposomes are self-assembling, colloidal particles in which a lipid bilayer, composed of amphiphilic molecules such as phosphatidyl serine or phosphatidyl choline, encapsulates a portion of the surrounding media such that the lipid bilayer surrounds a hydrophilic interior. Unila mellar or multilammellar liposomes can be constructed such that the interior contains a desired chemical, drug, or, as provide herein, an isolated nucleic acid molecule.

Liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells as well as mammalian cells. In order for a liposome to be an efficient gene transfer vehicle, characteristics among the following should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al, Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting uses the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted delivery system are ligands and receptors permitting the targeted delivery system to find and "home in" on the desired cells. A ligand may be any compound of interest that interacts with another compound, such as a receptor.

In general, surface membrane proteins that bind to specific effector molecules are referred to as receptors. Antibodies are preferred receptors. Antibodies can be used to target liposomes to specific cell-surface ligands. For example, certain antigens expressed specifically on tumor cells, referred to as tumor-associated antigens (TAAs), may be exploited for the purpose of targeting antibody-zinc finger-nucleotide binding protein-containing liposomes directly to the malignant tumor. Since the zinc finger-nucleotide binding protein gene product may be indiscriminate with respect to cell type in its action, a targeted delivery system offers a significant improvement over randomly injecting non-specific liposomes. A number of procedures can be used to covalently attach either polyclonal or monoclonal antibodies to a liposome bilayer. Antibody-targeted liposomes can include monoclonal or polyclonal antibodies or fragments thereof such as Fab, or F(ab')₂, as long as they bind efficiently to an the antigenic epitope on the target cells. Liposomes may also be targeted to cells expressing receptors for hormones or other serum factors. Delivery of constructs to cells

The cells may be transfected in vivo, ex vivo or in vitro. The cells may be transfected as primary cells isolated from a patient or a cell line derived from primary cells, and are not necessarily autologous to the patient to whom the cells are ultimately administered. Following ex vivo or in vitro transfection, the cells may be implanted into a host. Genetic modification of the cells may be accomplished using one or more techniques well known in the gene therapy field (see, e.g., (1994) Human Gene Therapy 5:543-563). Administration of a nucleic acid molecules provided herein to a target cell in vivo may be accomplished using any of a variety of techniques well known to those skilled in the art. The vectors of the herein may be administered orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and therefore melt in the rectum and release the drug.

The dosage regimen for treating a disorder or a disease with the vectors and/or compositions provided is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined empirically using standard methods.

The pharmaceutically active compounds (i.e., vectors or ligands) can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals. For oral administration, the pharmaceutical composition may be in the form of, for example, a capsule, a tablet, a suspension, or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of DNA or viral vector particles (collectively referred to as "vector"). For example, these may contain an amount of vector from about 10³-10¹⁵ viral vector particles, preferably from about 10⁶-10¹² viral particles. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods. The vector may also be administered by injection as a composition with suitable carriers including saline, dextrose, or water.

When a retroviral vector or lentiviral vector includes a polynucleotide encoding SCMl 13 protein or a variant, fragment, or derivative thereof is employed to administer the SCMl 13 protein or a variant, fragment, or derivative thereof to an animal, the vector particles are administered to the animal in an amount effective to promote angiogenesis in the animal. The animal may be a mammal, including human and non-human primates. Administration of these vector particles may be by systemic administration or by local injection. Examples of systemic administration include, but are not limited to, intravenous or intraarterial administration. A few nonlimitng examples of local injection include, but are not limited to, intramuscular, intratumoral or ocular injections including intraocular, subretinal and periocular injections, hi the case of systemic injections, the retroviral or lentiviral vectors are administered in an amoimt of at least 10⁴ transducing units (TU)/ml, and in general, such an amount does not exceed 10⁹ TU/ml. For local injections, the retroviral and lentiviral vectors are administered in an amount from about 10⁴ to 10⁷ TU per injection. Preferably for ocular applications, the retroviral or lentiviral vectors are administered in an amount of from about 10⁵ TU to about 10 TU per injection. The exact dosage to be administered is dependant upon a variety of factors, including the age, weight, and sex of the animal or patient to be treated. In the case of local injections, the retroviral vectors are administered in an amount of at least 10⁴ transducing units (TU), and in general, such an amount does not exceed 10⁹ TU. Preferably, the retroviral vectors are administered in an amount of from about 10⁵ TU to about 10⁷ TU. In the case of subretinal injection, the retroviral vectors are preferably administered in an amount of from about 10⁵ TU to about 10⁷ TU; most preferably from about 10⁵ TU to about 10⁶ TU. The exact dosage to be administered is dependant upon a variety of factors, including the age, weight, and sex of the animal or patient to be treated.

In another embodiment, an adenoviral vector is administered systemically in an amount from about 5xl0⁹ plaque forming units to about 5xl0¹³ particles per kg; more preferably from about 5xl0¹⁰ to about lxlO¹³ particles per kg; most preferably from about lxlO¹¹ to about 1x10 particles per kg. hi another embodiment, the adenoviral vector is administered by local injection at a dose of 10⁴ to 10¹³ particles per injection; preferably, the adenoviral vectors are locally administered in an amount of from about 10⁵ to about 10⁷ particles per injection. In the case of subretinal and similar injections, the adenoviral vectors are preferably administered in an amount of from about 10⁴ particles to about 10⁷ particles per injection; more preferably about 10⁵ particles to about 10⁷ particles per injection; most preferably about 10⁵ particles to about 10⁶particles per injection. In the case of intratumoral and similar injections, the adenoviral vectors are administered in an amount of from about lxlO⁶ to about lxlO¹³ particles per injection; preferably from about lxlO⁶ to about l lO¹¹ particles per injection; more preferably 5xl0⁹ to about lxl 0^π particles per injection. The exact dosage to be administered is dependant upon a variety of factors, including the age, weight, and sex of the animal or patient to be treated. In another embodiment, cells transduced with the adenoviral vector may be administered in an amount of from about 10³ to about 10⁸ cells, preferably from about 10⁴ cells to about 10⁸ cells, h general, the adenoviral vectors can be administered at the local site of ischemia or where therapeutic angiogenesis is required. Delivery can be performed by a variety of means including, but not limited to, direct injection of the adenoviral vector or cells transduced with the adenoviral vector, intraarterial delivery by a guided catheter or by computer guided systems such as NOGA, or by electroporation.

While the nucleic acids and /or vectors herein can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors or other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

Ligands similarly may be delivered by any suitable mode of administration, including by oral, parenteral, intravenous, intramuscular and other known routes. Any known pharmaceutical formulations is contemplated.

The following examples are to illustrate the invention, but should not be interpreted as a limitation thereon.

EXAMPLES The invention will be further described by reference to the following detailed examples.

These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by, for example, Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (2001); and by T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (1984).

Example 1 : cDNA Library Construction Following informed consent, human donors were treated with cyclophosphamide plus granulocyte-macrophage colony stimulating factor (GM-CSF) to mobilize CD34⁺ Thy-1⁺ hematopoietic stem cells (HSCs) to the peripheral blood. HSCs from multiple donors were combined. After apheresis, CD34⁺Thy-l⁺HSC stem cells were purified by flow sorting as described by Gazitt, et al., Blood, 86:381-389 (1995). Total RNA was purified from >10⁷ HSC using RNA-Stat (Tel-Test B Inc., Friendswood, Texas). PolyA⁺ RNA was purified from total RNA on oligo dT (Pharmacia Biotech) and used to synthesize cDNA (Stratagene unidirectional cDNA synthesis kit). Each cDNA molecule generated using this kit has an EcoRI sticky end at the 5' end and a Xhol sticky end at the 3' end. The cDNA was directionally cloned into lambda ZAP express that had been digested with EcoRI and Xhol restriction enzymes (Stratagene). The ligated cDNA/lambda ZAP was packaged using Gigapack III gold (Stratagene) and transfected into XLl-Blue MRF's cells (Stratagene). A total of 0.5 x 10⁶ independent clones were produced. The lambda phage were harvested and in vivo excised to pBlueScript (pBS) using ExAssist helper phage and SOLR strain E. coli according to recommended Stratagene protocol.

Random clones were mini prepped by Qiagen 96 well system, restriction digestion with EcoRI plus Xhol and electrophoresed to show inserts in the size range 0.5-5.0 Kb with an average size of 2.3 Kb. 10,000 mini prep clones were sequenced using T3 primed (i.e., 5' end) dye terminator sequencing reactions and processed on an ABI377 automated sequencer (PE Applied Biosystems). Sequence data was analyzed by BLASTX and BLASTN (Basic Local Alignment Search Tool) searches against GenBank. A number of clones were identified as being either completely novel or having homology only with ESTs.

Expression profiling was used to identify cDNA sequences which are preferentially expressed in HSCs. The cDNA inserts of clones identified as being either completely novel or having homology only with expressed sequence tags (ESTs) were amplified by PCR using T3 and T7 primers and then sent to Synteni where the microdot arrays were generated. Microdot array probes were synthesized from RNA purified from mobilized peripheral blood CD34 cells and labeled with Cy3 and from either peripheral blood cells (PBL) RNA or GDI lb RNA or CD4 RNA or CD 19 RNA and labeled with Cy5 using standard protocols as recommended by Synteni. The CD34 probe and the PBL probe were mixed and allowed to hybridize to a microdot array. After hybridization and washing, the microarray was scanned to determine the intensity of probe binding to each cDNA. Hybridizations, washing and scanning were performed by Synteni. Probe binding is proportional to gene expression level. The raw binding data was balanced by monitoring probe binding to Synteni control elements on the microarray; this accounts for differences in the fluorescent labeling of the two probes. The ratio of the two binding intensities, the balanced differential expression (BDE) gives a quantitative measurement of relative gene expression level.

Analysis allowed the identification of 101 new cDNAs that were expressed more in stem cells than in peripheral blood cells (PBL), these cDNAs were designated selected cDNAs. The selected cDNAs are defined as being expressed at least two-fold higher in stem cells (BDE>2.0) and have a low expression in PBL cells. The control cDNAs, CD34, flk2 (fetal liver kinase) and KIT (stem cell factor or alternatively steel factor, or c-Kit ligand) are known to be preferentially expressed in HSC and this is confirmed using transcript imaging. The BDE for SCMl 13 was 2.7. The increased expression in CD34+ cells was verified by Northern blotting analysis where a 2 to 3 fold increase in messenger RNA levels also was observed.

Two approaches were taken to prioritize the 101 selected cDNAs: sequence analysis was used to confirm their new classification and further transcript imaging experiments were performed to investigate levels of expression in subsets of peripheral blood cells. Microdot arrays were analyzed with probes specific for CD34⁺ cells compared with either T cells (CD3*), B cells (CD19 ) or myeloid cells (GDI lb^"1). High priority cDNAs were confirmed to be novel and had MSC-restricted expression (i.e., relatively low expression in PBL, B, T and myeloid cells). The expression pattern of SCMl 13 met the above criteria. The cDNA insert in SCMl 13 corresponds to SEQ ID NO: 1.

Example 2: Vector Construction cDNA inserts were subcloned from pBS and into an MSCV based retroviral vector (Hawley et al., Gene Therapy, 1:136-138 (1994). The cDNA inserts were subcloned into vector MIE (see Figure 1). MIE was constructed from MINGFR (Cheng et al., Blood 92:83-92 (1998) by removing the nerve growth factor receptor (NGFR) gene and replacing it with enhanced green fluorescent protein (EGFP) gene on a 707bp Ncol - blunted Bspl fragment. The NGFR gene was replaced by restriction digestion with Clal, filling in the sticky end and then digestion with Ncol. The EGFP was isolated from pEGFP-1 (Clontech) and has GenBank Accession No. U55761. MIE vector has the essential components LTR-IRES-EGFP. The cDNA insert is cloned into MIE at the EcoRI site by PCR of the coding region of SCMl 13 and cloning to PCR2, removal from PCR2 by EcoRI digestion and ligation into MIE. This gives gene expression mediated by the LTR and the ribosome entry site (IRES) allows for simultaneous translation of both the gene of interest and EGFP proteins from one primary transcript. Expression of EGFP allowed selection of transduced cells by FACS .

The SCMl 13 cDNA fragment containing the entire coding region of SEQ HD NO:l was amplified by PCR and the 3' primer included an in-frame hemagglutinin (HA) tag (5' TAC CCC TAC GAC GTG CCC GAC TAC GCC - SEQ ID NO:3) followed by a stop codon, was subcloned into the MIE vector at the EcoRI site. The HA tag and anti-HA antibodies were used to follow protein expression by Western Blots. Example 3: Retroviral Infection

The retrovirus was produced by transfecting retroviral vector into the RV packaging cell line phoenix (Kinsella et al., Human Gene Therapy, 7(12):1405-1413, 1996) obtained from Nolan Laboratories using a standard transfection protocol (Clontech cat. #K20517-1). Viral supematants were harvested 12, 24, 36, and 48 hours after transfection.

Example 4: Overexpression of SCMl 13 Gene in Endothelial Cells To deliver the SCMl 13 gene into human umbilical vein endothelial cells (HUVEC) (Clonetics cat. no. CC2519), cells in complete growth media (Clonetics cat. no. CC-3125) were transduced with retroviral vectors SCMl 13 or MIE by spinoculation (2,400g) for 3 hours in the presence of 8μg/ml of Polybrene. After spinoculation, cells were cultured in fresh growth media for 48 to 72 hours. Cells expressing EGFP protein were selected by fluorescence activated cell sorting (FACS) and used for further assays. On average, the transduction efficiency ranged from 30 to 80%. Retroviral transduction resulted in constitutive stable expression of EGFP protein. This was confirmed by FACS analysis of cells (EGFP expression) following extended culture.

Example 5: SCMl 13 Increases Migration of HUVEC Cells

Endothelial cells can migrate towards angiogenic growth factors or chemotactants. In vitro cell migration was measured by two different methods. The "tilt assay" is a recently developed assay. HUVEC were suspended in EBM (a basal medium) and seeded in one-half of each well of a 12-well plate. The plate was tilted to an angle of 50-60°, incubated for 12-18 hours and then placed horizontally. Media with growth factors (FGF or VEGF) was added. At this point, half of each dish contained a confluent monolayer of cells and the other half was cell-free. During 6-8 hours of incubation, cells migrated into the empty half of each well. After 6-8 hours, cells were fixed with methanol and subsequently stained by Giemsa stain. The cells that migrated into the empty half of each dish were counted. With this assay, a linear dose- response of HUVEC migration was established in the presence of 1-10 ng/ml human FGF (Figure 2). Overexpression of SCMl 13 in HUVEC cells resulted in a 5-fold increased cell migration in response to 25 ng/ml of VEGF over control cells (cells expressing MIE vector). Similarly, in the 2 ng/ml of FGF, a 2.5 - 5-fold increase of cell migration in SCMl 13 transduced cells was determined (Figure 3).

To confirm increased endothelial cell migration due to SCMl 13 overexpression, a transwell migration assay was performed with SCMl 13 transduced cells. Transwell chambers (Corning cat. #3472) were coated with diluted 1 :20 matrigel (BD cat. # 356234) at 37°C for 1 hour. 1 x 10⁵ transduced HUVEC resuspended in EBM with 5% FBS were plated onto the insert. 0.5 ml of media with 10 ng FGF was added into the bottom section of the transwell. After 6-8 hours of incubation at 37°C, 5% CO₂, nonmigrated cells were removed from the top chamber. Migrated cells on the bottom insert were fixed with cold methanol and subjected to Giemsa staining. The numbers of migrated cells were determined by counting using an inverted microscope. As shown in Figure 4, the migration of the SCMl 13 transduced HUVEC was 3-5 fold greater than control cells.

Example 6: SCMl 13 overexpression result in enhanced endothelial cell proliferation in response to angiogenic growth factors

We have also analyzed the cell proliferation of HUVEC in the presence of overexpressed SCMl 13 by ³H-thymidine incorporation method. The amount of incorporated to cells ³H-thymidine (cpm) is proportional directly to the amount of DNA synthesized or the number of cells. (Janat, et al., J. Cell. Physiol., Vol. 150, pgs. 232-242 (1992)). Cells were seeded in 96 well plate at 2,500-5,000 cell/well, and cultured in basal medium (EBM, R&D) + 5% FBS for 48 hours. Then, media with or without growth factors, e.g., human basic rFGF (R&D system cat. #233-FB) or human rVEGF (R&D system cat. #298-VS) were added. Cell culture was continued for another 24 hours. At 6 hours before harvesting the cells, 0.5 uCi ³H- thymidine was added to each sample according to manufacturer's recommendations. Incorporation of ³H-Thymidine was counted by scintillation counter. Samples were set in triplicates for each condition in each experiment. We have tested the effect of the SCMl 13 gene on thymidine incorporation using different concentrations of growth factors (see Table 1 below). Overexpression of SCMl 13 resulted in a consistent increase (up to 50%) in the amount of thymidine incorporation.

Table 1

3H-Thymidine incorporation (cpm)

Growth Factors SCM113 MIE

FGF (0.25ng/ml) 7077+509 5243+336

FGF (lng/ml) 7209+689 6482+413

VEGF (lOng/ml) 6334+761 4747+334

VEGF (20ng/ml) 6282+413 5185+205

VEGF (25ng/ml) 10,476+1202 6943+875 bovine brain extract 7459+462 5924+555

Cell proliferation of transduced HUVECs in response to growth factors Data are expressed as mean + SD from triplicate samples. Example 7: SCMl 13 overexpression result in an elongated endothelial cell shape and an enhanced morphogenic response

We noted that the morphology of endothelial cells overexpressing SCMl 13 were elongated considerably. The cells exhibited elongated morphology in sparse culture and formed tubule-like structures with increased confluency. Furthermore, in denser cultures and during cell migration assays we detected increased formation of tube-like structures in culture. The data suggest that in addition to increased cell migration and proliferation, SCMl 13 overexpression enhances the formation of capillary-like networks. These in vitro activities are good predictors of angiogenesis in vivo and our data support an important role for SCMl 13 as an important and novel modulators of angiogenesis.

It is to be understood that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.The disclosures of all patents, publications (including published patent applications), depository accession numbers, and database accession numbers are herein incorporated by reference to the same extent as if each patent, publication, depository accession number, and database accession number were specifically and individually incorporated by reference.

Claims

What Is Claimed Is:

I . A method for promoting neovascularization in an animal comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to promote neovascularization in said animal.

2. A method for preventing or treating congestive heart failure in an animal, comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to prevent or treat congestive heart failure in said animal.

3. A method for preventing or treating myocardial ischemia in an animal comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to prevent or treat myocardial ischemia in said animal.

4. A method for treating ischemia-reperfusion injury in an animal comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to treat ischemia-reperfusion injury in said animal.

5. A method for treating a peripheral arterial disease in an animal comprising administering to said animal SCMl 13 protein, or a variant, fragment, or derivative thereof in an amount effective to treat the peripheral arterial disease in said animal.

6. The method of any one of claims 1-5, wherein said SCM113 protein, or variant, fragment, or derivative thereof comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2.

7. The method of any one of claims 1-5, wherein said SCM113 protein, or variant, fragment, or derivative thereof comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2.

8. The method of any one of claims 1-5, wherein said SCMl 13 protein, or variant, fragment, or derivative thereof comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:2.

9. The method of any one of claims 1-5, comprising administering to said animal SCMl 13 protein comprising an amino acid sequence as shown in SEQ ID NO:2 or an active fragment thereof.

10. The method of any one of claims 1-5, wherein said SCMl 13 protein, or variant, fragment, or derivative thereof is administered to said animal as a recombinant protein.

I I . The method of claim 10, wherein said protein is administered orally, intravenously, intramuscularly, intraperitoneally, or intrathecally.

12. The method of any one of claims 1-5, wherein said SCM113 protein, or variant, fragment, or derivative thereof is administered by administering a vector comprising a nucleotide sequence encoding said SCMl 13 protein, or variant, fragment, or derivative thereof.

13. The method of claim 12, wherein said vector is a plasmid, a lipid formulation, or a viral vector.

14. The method of claim 13, wherein said vector is administered orally, intravenously, intramuscularly, intraperitoneally, or intrathecally.

15. The method of any one of claims 1-5, wherein said SCM113 protein, or variant, fragment, or derivative is administered to said animal by transducing cells of blood vessels with an expression vehicle comprising a gene construct encoding said SCMl 13 protein, or analogue, fragment or derivative thereof.

16. The method of claim 13 wherein said vector is a viral vector.

17. The method of claim 16, wherein said viral vector is an adeno associated viral vector, a plasmid, or an adenoviral vector.

18. The method of claim 16, wherein said viral vector is an adenoviral vector.

19. The method of claim 16, wherein said viral vector is a retroviral vector.

20. The method of claim 19, wherein said retroviral vector is a MoMLV-vector

21. The method of claim 16, wherein said viral vector is a lentiviral vector.

22. The method of claim 21, wherein said lentiviral vector is derived from a lentivirus selected from the group consisting of HIV, BIV, EIAV, FIV, and SIV.

23. The method of claim 12, wherein said nucleotide sequence encoding said SCMl 13 protein, or variant, fragment, or derivative thereof is at least 85% identical to SEQ ID NO:l.

24. The method of claim 12, wherein said nucleotide sequence encoding said SCMl 13 protein, or variant, fragment, or derivative thereof is at least 90% identical to SEQ ID NO:l.

25. The method of claim 12, wherein said nucleotide sequence encoding said SCMl 13 protein, or variant, fragment, or derivative thereof is at least 95% identical to SEQ ID NO:l.

26. The method of claim 12, wherein said nucleotide sequence comprises SEQ ID NO: 1.

27. The method of claim 12, wherein said vector further comprises a nucleotide sequence encoding a protein selected from the group consisting of VEGF, FGF, IGF, an angiopoietin, PD-EGF, TGF-/3, fflFl-α, nitric oxide synthase, MCP-1, Lιterleukin-8, ephrins, NAP-2, ENA- 78, GROW- a, and an active fragment of tyrosyl-tRNA synthase.

28. The method of claim 18, wherein said adenoviral vector is administered to said animal in an amount of from about 5xl0⁹ plaque forming units to about 5x10¹³ particles per kilogram.

29. The method of claim 18, wherein said adenoviral vector is administered to said animal in an amount of from about 5xl0¹⁰ plaque forming units to about lxlO¹³ plaque forming units.

30. The method of claim 18, wherein said adenoviral vector is administered to said animal in an amount of from about lxlO¹¹ transducing units to about 10¹³ transducing units.

31. The method of claim 21 , wherein said lentiviral vector is administered to said animal in an amount of from about lxlO⁴ transducing units to about 10¹¹ transducing units.

32. The method of claim 21, wherein said lentiviral vector is administered to said animal in an amount of from about 1x10 transducing units to about 1x10 transducing units.

33. The method of claim 21 , wherein said lentiviral vector is administered to said animal in an amount of from about lxlO⁵ transducing units to about lxlO⁶ transducing units.

34. The method of any one of claims 1-5, wherein said animal is a mammal.

35. The method of claim 34, wherein said mammal is a primate.

36. The method of claim 35, wherein said primate is a human.