AAV VECTOR-BASED COMPOSITIONS AND THERAPEUTIC USES THEREOF

Abstract: Disclosed are methods for the use of cytokine or cytokine receptor-encoding polynucleotides in the creation of transformed host cells and transgenic animals is disclosed. In particular, the use of recombinant adeno-associated viral (rAAV) vector compositions comprising polynucleotide sequences that express one or more mammalian cytokines or cytokine receptor polypeptides is described. Also disclosed are methods for the treatment and amelioration of symptoms of a variety of conditions and disorders in an animal, including blindness, retinitis pigmentosa, age-related macular degeneration, obesity, anorexia, and related eating disorders, as well as neurological and musculoskeletal disorders, including for example, amyotrophic lateral sclerosis.

Description: DESCRIPTION AAV VECTOR-BASED COMPOSITIONS AND THERAPEUTIC USES THEREOF 1.0 BACKGROUND OF THE INVENTION The present application claims priority to United States serial number 09/138,226, filed June 9, 1999, the entire contents of which is specifically incorporated herein by reference in its entirety. The United States government has certain rights in the present mvention pursuant to Grant Numbers NS32727, DK37273 and GM53252 from the National Institutes of Health. 1.1 FIELD OF THE INVENTION The present invention relates generally to the fields of molecular biology and virology, and in particular, to methods for using recombinant adeno-associated virus (rAAV) compositions that express nucleic acid segments encoding therapeutic gene products in the treatment of complex human disorders. In certain embodiments, the invention concerns the use of rAAV in a variety of investigative, diagnostic and therapeutic regimens, including the treatment of diseases of the nervous and musculoskeletal systems, blindness, age-related macular degeneration, obesity, weight gain, and various eating disorders. Methods and compositions are provided for preparing rAAN-based vector constructs that express one or more cytokine or cytokine receptor gene(s) for use in viral-based gene therapies. 1.2 DESCRIPTION OF THE RELATED ART An estimated 54 percent of adults in the United States are overweight, according to the National Institutes of Health. Treating obesity and its related conditions, including high blood pressure, heart disease and diabetes, is estimated to cost more than $45 billion annually, according to an August, 1996 Scientific American article. With the incidence of obesity on the rise in the United States, there is a need to develop therapies that will alleviate the symptoms associated with obesity. Two appetite-controlling compounds have been previously identified in mammals- leptin and ciliary neurotrophic factor (CNTF). Leptin is a naturally occurring protein produced by fat cells that inhibits appetite and increases energy expenditure. It signals the brain, affecting the brain's secretion of appetite-regulating signals. Such signals include neuropeptide Y, a chemical that has been found to stimulate appetite. This process is thought to be faulty in most obese people so that even high levels of leptin fail to turn off the hunger signal. LIF is a secreted polyfunctional cytokine that elicits a diversity of biological effects on many cell types. LIF's action is mediated following binding to specific cellular receptors that trigger differentiation-induction, differentiation-suppression, proliferation, and activation depending on the cell type. The distribution of LIF receptor (LIFR) mRNA in the adult brain and spinal cord suggests that LIF or LIF related cytokine exert important actions on the neuronal cells of the central nervous system in the adult as well as during development. LIFRR mRNA expression is largely restricted to specific brain regions relevant to the motor and sensory systems, and in the spinal cord expression is largely found in the motor neurons of the ventral hom and in the sensory ganglia. 1.3 DEFICIENCIES IN THE PRIOR ART Currently, there are limited pharmacological approaches to treating eating disorders, obesity, neurological dysfunction, and musculoskeletal disorders and diseases in an affected animal. Many such methods introduce undesirable side-effects, and do not overcome the problems associated with traditional modalities and treatment regimens for conditions such as retinitis pigmentosa, age-related macular degeneration, age-related muscle weakness, amyotrophic lateral sclerosis, and the like. Thus, the need exists for an effective treatment that circumvents the adverse effects and provides more desirable results, with longer acting effects, and improved patient compliance. In addition, methods for delivery of polynucleotides to a host cell that express a cytokine or cytokine receptor polypeptide useful in the amelioration of such conditions, and in particular, administration of specific rAAN-based polynucleotide constructs to a mammal are particularly desirable. 2.0 SUMMARY OF THE INVENTION The present invention overcomes these and other limitations inherent in the prior art by providing new rAAN-based genetic constructs that encode one or more mammalian cytokines or cytokine receptor polypeptides for the treatment or amelioration of various disorders resulting from a cytokine or cytokine receptor polypeptide deficiency. In particular, the invention provides genetic constructs encoding one or more mammalian cytokine or cytokine receptor polypeptides, for use in the treatment of such conditions as blindness, retinitis pigmintosa, age-related macular degeneration, obesity, anorexia, weight gain, and a variety of eating disorders. Likewise, the invention provides genetic constructs that encode one or more cytokines or cytokine receptor polypeptides useful in the treatment or amelioration of various neuromuscular disorders, including for example, amyotrophic lateral sclerosis, and related conditions that manifest from a deficiency or absence of physiologically normal levels of cytokines or cytokine receptor polypeptides. The invention provides compositions and methods for treating or ameliorating such a cytokine-or a cytokine receptor polypeptide deficiency in a mammal, and particularly for treating or reducing the severity or extent of deficiency in a human manifesting one or more of the disorders linked to a deficiency in such polypeptides. In a general sense, the method involves administration of an rAAV-based genetic construct that encodes one or more anorectic cytokines or cytokine receptor polypeptides in a pharmaceutically-acceptable vehicle to the animal in an amount and for a period of time sufficient to treat or ameliorate the deficiency in the animal suspected of suffering from such a disorder. Exemplary cytokines useful in the practice of include, but are not limited to those described herein in Table 4, and include polypeptides such as leptin (Lep), BDNF, LIF, BDNF receptor, LIF receptor, leptin receptor, ciliary neurotrophic factor (CNTF), and CNTF receptor polypeptide. In one embodiment, the cytokine or cytokine receptor polypeptide deficiency manifests itself in musculoskeletal dynfunction. In such instances, the invention provides a method for inhibiting, reducing, or ameliorating age-related muscle weakness or muscle fatigue in a mammal, and preferably a human. The method generally involves administering to the mammal an effective amount of an rAAV composition that comprises a selected polynucleotide sequence encoding a mammalian cytokine or cytokine receptor polypeptide to inhibit, reduce, or ameliorate the muscle dysfunction, weakness or muscle fatigue in the mammal. In a second embodiment, the cytokine or cytokine receptor polypeptide deficiency manifests itself in a neuromuscular disease or disorder. In such instances, the invention provides a method for treating or reducing the severity of symptoms of the neuromuscular disease in an animal. This method generally involves administering to an animal suspected of having one or more neuromuscular diseases or disorders a therapeutically-effective amount of an rAAN vector construct that comprises a DΝA segment that encodes at least a first mammalian cytokine or cytokine receptor polypeptide in an amount and for a time effective to treat or ameliorate such a neuromuscular disease. In a third embodiment, the cytokine or cytokine receptor polypeptide deficiency manifests itself in an age-related muscle deterioration. In such instances, the invention provides a method for treating, ameliorating, or reducing the severity of symptoms of one or more types of age-related muscle deterioration in a mammal. This method generally involves administering to the mammal affected with such a condition an amount of an rAAV vector construct that comprises a selected polynucleotide sequence encoding at least a first mammalian cytokine or cytokine receptor polypeptide effective to treat, ameliorate or reduce the severity of symptoms of such muscle deterioration or dysfunction in the mammal. In a fourth embodiment, the cytokine or cytokine receptor polypeptide deficiency manifests itself in the form of visual impairment, blindness, retinitis pigmentos, or age- realted macular degeneration. In such instances, the invention provides a method for treating, ameliorating, or reducing the severity of symptoms of one or more defects in vision. The administration of one or more such cytokine or cytokine receptor polypeptide genetic constructs is preferred in treating such ophthalmic conditions that develop or are worsened by a deficiency of cytokine or cytokine receptor polypeptides. 3.0 BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which: FIG. 1A shows that body weight was significantly (p<0.05) reduced by a single 50 μl intravenous injection of rAAV-leptin in obese male o o ] j mice (50-60 g) in a dose dependent manner, the highest dose being the most effective; FIG. IB shows that food intake in ob/ob mice following rAAV-leptin was also decreased by the highest dose (p<0.05 vs control); FIG. 1C shows that serum leptin levels in ob/ob m * ce were elevated significantly after intravenous injection of 10 11 particles of rAAV-leptin; FIG. 2 shows representative 0 b/ 0 b mice control vs rAAV-Leptin treated; FIG. 3A shows that body weight was significantly reduced by 2 weeks post injection in rAAV-leptin-treated SD rats as compared with control rats; this reduction was maintained for the duration of the study (*= p <0.05); FIG. 3B shows that food intake in rAAV-leptin vs control SD rats. There was no change in overall food intake between the control and experimental groups; FIG. 3C shows that serum leptin levels were significantly reduced in rAAV-Leptin treated SD rats (0.08 ± 0.01 ng/ml) VΛ controls (2.2 ± 0.42 ng/ml); FIG. 4 shows the effects of icr rAVV-CNTF in SD rats (p<0.05 vs rAAV-GFP controls); FIG. 5 shows the effects of icr rAW-CNTF in SD rats (p<0.05 vs rAAV-GFP controls); FIG. 6A shows the effect of icv rAAV-Leptin in 24 day old SD rats (p<0.05 vs _ rAAV- GFP injected controls at corresponding time points); FIG. 6B shows the effect of icv rAAV-Leptin in 24 day old SD rats (p<0.05 VΛ rAAV- GFP injected controls at corresponding time points); FIG. 7 shows the effect of rAAV-Leptin on UCP-l mRNA levels in interscapular Brown adipose tissue. UCP-l mRNA levels were significantly upregulated in rAAV-leptin treated rats (9) vs control (9)at 6 weeks post injection; FIG. 8 shows the effect of rAAV-Leptin on body fat composition. There was significant reduction in body fat (g) levels (p<.05) in rAAV-leptin treated animals (n=3) vs control (11=3) at 6 weeks post injection; FIG. 9 shows the effect of rAAV-Leptin on protein composition. There was no change in protein levels with <.05 in rAAV-leptin treated animals (n=3) vs control (n=3) at 6 weeks post injection (p<0.5 p<.05); FIG. 10 shows the effect of icv injections of leptin and CNTF on STAT-3 and STAT-1 phosphorylation in hypothalamus of rats. Recombinant rat leptin (R&D Systems; 5μg in 5M1 of PBS) and CNTF (A gen; 5 μ g in 5 μ l of PBS) were injected icv to SD male rats (250-300g). Animals were sacrificed 15 and 45 min. post-injection. Protein extracts of hypothalamus were Western blotted with α STAT-3, α P(Tyr-705)STAT-3, α P(Ser701)STAT-3, α STAT-l, or α P(Tyr701)STAT-l after SDS-PAGE (7.5%). The representative immunolots are shown. The intensities of bands of phosphorylated STATs obtained by image analysis were normalized to corresponding total STATs levels and expressed as arbitrary units; each bar is mean + SEM for 3-5 animals for each time point; *, PO.01 (t-test) vs . corresponding control; FIG. 11A shows a diagram of rAAV-CNTF; FIG. 11B shows a nucleotide sequence coding for hGH signal peptide. The codons of the hGH are shown in italic, the first two codons of the human CNTF cDNA are shown in bold uppercase, the consensus Kozak sequence is underlined; FIG. 12 shows a diagram of the rAAN-Ob vector; FIG. 13 shows the diagram of rAAV vectors designed and constructed for the current project. IRES is a poliovirus type 1 internal ribosome entry site that mediates a coordinate expression of a transgene and GFP reporter gene from dicistronic transcription unit; FIG. 14 shows the effect of CNTF gene therapy on ONL thickness in the rds +/- Tg P216L mouse using various rAAV constructs. ONL thickness was determined at 21 regular intervals along a full vertical meridian through the optic nerve head (ONH) 75 days postinjection (P90). The left half is inferior retina; the right half is superior retina. The number of eyes averaged for each treatment is indicated; and FIG. 15 shows a schematic that indicates pathways affected by hypothalamus appetite controlling signals and the relation of various inhibiting and stimulatory signals to gut response and leptin release. 4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure. Using a mammalian animal model, the inventors have demonstrated the beneficial effects of these constructs and their efficacy of anorexigenic and body weight (BW) reducing effects. As an illustrative example, an rAAV construct comprising a gene encoding leptin (rAAV-leptin) delivered either intramuscularly m ) or intravenously ( v ) reduced BW in leptin-deficient obese 0 b/ob mice. rAAV-leptin injected v produced a dose-dependent decrease in BW concurrent with reduction in food intake (FI). In lean Sprague-Dawley (SD) rats, rAAV-leptin delivered intracerebroventricularly ( cv ) also suppressed the normal BW gain. In a second illustrative embodiment, the efficacy of rAAV-introduced CNTF-encoding polynucleotides was demonstrated in leptin-resistant genetically obese Zucker (fa/fa) rats. An rAAV-based genetic construct encoding a secreted form of CNTF was administered (10 10 physical particles) j cv to f a /f a rats. The results showed that rAAV-CNTF decreased the rate of BW gain accompanied by a significant reduction in cumulative FI. In a third illustrative embodiment, the relative efficacy of other related cytokines to regulate BW after peripheral or central administration using rAAV vectors have been demonstrated. These results demonstrate that cytokine and cytokine receptor-encoding gene therapy using an rAAV delivery system is a viable alternative to pharmacologic approaches for reducing body weight for extended periods of time. 4.1 OBESITY Obesity is a complex disorder and often leads to hyperinsulinemia, hyperglycemia and insulin resistance. Obesity is also a major risk factor for hypertension and cardiovascular disease. There are multiple pathways controlling the complex balance of energy intake and expenditure. The major afferent factor in a negative feedback loop regulating daily food intake and body weight is hormone leptin synthesized in adipocytes. Although leptin administration has been shown to reduce food intake and body weight in rodents its effectiveness is only transient and requires repeated injections. In humans, plasma levels of leptin increase in direct correlation with increase in body weight and adiposity; this tolerance to leptin (leptin resistance) is believed to be an underlying factor in the loss of leptin control on energy balance. In addition, leptin-resistance due to environmental and genetic factors contributes substantially to human obesity. The cellular and molecule mechanisms of leptin resistance are not known. It is believed that leptin resistance is heterogeneous and multiple factors, including defective transport to brain and defective influence on the activity of the neural circuits that regulate body weight, are major players. Consequently, there is a clear need to develop innovative approaches to control appetite and body weight and correct obesity. Leptin belongs to a cytokine family of peptides. Several members of cytokines, e g ciliary neurotrophic factor (CNTF), are potent suppressors of appetite and body weight gain, and their efficacy has been demonstrated in normal and genetically obese rodent models. . These cytokines are the naturally occurring appetite suppressing molecules (anorexigenic), potentially useful for circumventing leptin-resistance and to serve as therapeutic agents not only to correct obesity but also to either prevent or maintain body weight at clinically appropriate ranges in normal and obese patient populations. 4.2 AMYOTROPHIC LATERAL SCLEROSIS (ALS) ALS is a chronic progressive disease of unknown disease of unknown etiology that results in the selective destruction of spinal cord, brainstem and cortical motor neurons, with patients typically dying within 3 years of disease onset. At present tliere are no effective therapies to halt or even slow the progression of the disease. The cause of ALS remains unknown although there is a general consensus that its multifactorial with factors and cytokines such as LIF or CNTF may prevent or reduce motor neuron cell death and slow down disease progression. Unlike the prior art, in which phase II-III clinical trials using subcutaneously administered recombinant human CNTF failed to demonstrate slowing disease progression in ALS patients, the present mvention provides tonic expression of a cytokine that is delivered by an rAAV expression system. This system overcomes the limitations in the prior art by reducing motor neuron cell death and slowing the progression of this degenerative disease. 4.3 AGE-RELATED MUSCLE WEAKNESS It has been demonstrated that subcutaneous administration of CNTF in aged 24-month old rats increased the muscle strength 2.5-fold. Consistent with this finding LIF administered by single f m injection to reinnervated muscle in adult rats resulted in an increase in muscle fiber diameter. The present invention provides improved methods for delivering cytokines, such as CNTF and LIF, using rAAV vector system that are effective therapeutic factors in treatment of age-related muscle weakness. 4.4 BLINDNESS In the human disease retinitis pigmentosa (RP) and age-related macular degeneration (AMD) blindness results from photorecptors degenerating v f a apoptic pathways due to a wide variety of genetic defects in a wide variety of genes expressed either in photoreceptors themselves or in neighboring cells supporting photoreceptor function. Many cytokines, particularly the neurotrophins, have been shown to slow this process of cell death, but singular intracular injections of cytokine proteins have not proven effective at the long-term protection required for RP and AMD. The present invention utilizes an rAAV system to deliver specific cytokine-encoding genes or cytokine receptor-encoding genes under regulation of either general promoter elements or photoreceptor specific promoter elements to ocular tissues to provide gene expression in the retina. This leads to a long-term continuous release of the passenger cytokine and prolonged photorecptor rescue. Animal model studies of RP have shown that gene transfer of ciliary neurotrophic factor (CNTF) or brain-derive neurotrophic factor (BDNF) using either the CMV or a proximal murine rod opsin (MOPS) promoter leads to rescue of vision for at least 3 months. Because cytokine receptor density may be low on many retinal cell types, analogous cytokine receptor gene delivery, either in isolation or combined with the cytokine gene itself, further enhances this rescue, and provides a superior method for the treatment of such disorders in mammals, and in particular, humans. 4.5 ADENO-ASSOCIATED VIRUS Adeno-associated virus-2 (AAV) is a human parvovirus that can be propagated both as a lytic virus and as a provirus (Cukor e t a h 1984; Hoggan e t a 1972). The viral genome consists of linear single-stranded DNA (Rose e tah 1969), 4679 bases long (Srivastava e t a L > 1983), flanked by inverted terminal repeats of 145 bases (Lusby e f a \ , 1982). For lytic growth AAV requires co-infection with a helper virus. Either adenovirus (Atchinson e f a \ , 1965; Hoggan, 1965; Parks e tal > 1967) or herpes simplex (Buller e t a , 1981) can supply helper function. Without helper, there is no evidence of AAV-specific replication or gene expression (Rose and Koczot, 1972; Carter e a , 1983). When no helper is available, AAV can persist as an integrated provirus (Hoggan, 1965; Berns e tal. ι 1975; Handa e f a \ , 1977; Cheung e t a L > 1980; ems et aL , 1982). Integration apparently involves recombination between AAV termini and host sequences and most of the AAV sequences remain intact in the provirus. The ability of AAV to integrate into host DNA is apparently an inherent strategy for insuring the survival of AAV sequences in the absence of the helper virus. When cells carrying an AAV provirus are subsequently superinfected with a helper, the integrated AAV genome is rescued and a productive lytic cycle occurs (Hoggan, 1965). AAV sequences cloned into prokaryotic plasmids are infectious (Samulski et a j , 1982). For example, when the wild type AAV/pBR322 plasmid, pSM620, is transfected into human cells in the presence of adenovirus, the AAV sequences are rescued from the plasmid and a normal AAV lytic cycle ensues (Samulski et a \ , 1982). This renders it possible to modify the AAV sequences in the recombinant plasmid and, then, to grow a viral stock of the mutant by transfecting the plasmid into human cells (Samulski et a \ , 1983; Hermonat et a , 1984). AAV contains at least three phenotypically distinct regions (Hermonat e f a j ., 1984). The re „ region codes for one or more proteins that are required for DNA replication and for rescue from the recombinant plasmid, while the CC φ and //« regions appear to code for AAV capsid proteins and mutants within these regions are capable of DNA replication (Hermonat e tal. > 1984). It has been shown that the AAV termini are required for DNA replication (Samulski e f a l, 1983). Laughlin e f a j (1983) have described the construction of two β co jj hybrid plasmids, each of which contains the entire DNA genome of AAV, and the transfection of the recombinant DNAs into human cell lines in the presence of helper adenovirus to successfully rescue and replicate the AAV genome (See also Tratschin e f a _, 1984a; 1984b). Adeno-associated virus (AAV) is particularly attractive for gene transfer because it does not induce any pathogenic response and can integrate into the host cellular chromosome (Kotin e t al, 1990). The AAV terminal repeats (TRs) are the only essential ^-components for the chromosomal integration (Muzyczka and McLaughin, 1988). These TRs are reported to have promoter activity (Flotte e f a f, 1993). They may promote efficient gene transfer from the cytoplasm to the nucleus or increase the stability of plasmid DNA and enable longer-lasting gene expression (Bartlett and Samulski, 1998). Studies using recombinant plasmid DNAs containing AAV TRs have attracted considerable interest. AAV-based plasmids have been shown to drive higher and longer transgene expression than the identical plasmids lacking the TRs of AAV in most cell types (Philip et α /., 1994; Shafron et a ι, 1998; Wang et a ι, 1998). . There are several factors that prompted researchers to study the possibility of using rAAV as an expression vector. One is that the requirements for delivering a gene to integrate into the host chromosome are surprisingly few. It is necessary to have the 145-bp ITRs, which are only 6% of the AAV genome. This leaves room in the vector to assemble a 4.5-kb DNA insertion. While this carrying capacity may prevent the AAV from delivering large genes, it is amply suited for delivering the antisense constructs of the present invention. AAV is also a good choice of delivery vehicles due to its safety. There is a relatively complicated rescue mechanism: not only wild type adenovirus but also AAV genes are required to mobilize rAAV. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimizes immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response. AAV therefore, represents an ideal candidate for delivery of the polynucleotides or hammerhead ribozyme constructs of the present invention. 4.6 PROMOTERS AND ENHANCERS Recombinant vectors form important aspects of the present invention. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In preferred embodiments, expression only includes transcription of the nucleic acid, for example, to generate a cytokine polypeptide product from a transcribed gene. Particularly useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. In preferred embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a cytokine-encoding gene in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the cytokine-encoding DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high-level expression of the introduced DNA segment. At least one module in a promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription. The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter, such as a CMV or an HSV promoter. In certain aspects of the invention, tetracycline controlled promoters are contemplated. In various other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes. The use of other viral or mammalian cellular or bacterial phage promoters that are well known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose. Tables 1 and 2 below list several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of the present cytokine-encoding constructs. This list is not intended to be exhaustive of all the possible elements involved in the promotion of transgene expression but, merely, to be exemplary thereof. Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. TABLE 1 PROMOTER AND ENHANCER ELEMENTS PROMOTER/ENHANCER REFERENCES lmmunogiobulin Heavy Chain Banerji et a t 1 83; Gilles e f a ι, 1 83; Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986, 1987; Imler et a 1987; Weinberger ^/., 1984; Kiledjian et a ., 1988; Porton ^ a /.; 1990 lmmunogiobulin Light Chain Queen and Baltimore, 1983; Picard and Schaff er, 1984 T-Cell Receptor Luria et a \ , 1987; Winoto and Baltimore, 1989; Redondo et ai:Λ99) HLA DQ a and DQ β Sullivan and Peterlin, 1987 β-Interferon Goodbourn e f a \ , 1986; Fujita e f a \ , 1987; Goodbourn and Maniatis, 1988 Interleukin-2 Greene et a ι, 1989 Interleukin-2 Receptor Greene et aL , 1989; Lin et aL , 1990 MHC Class II 5 Koch et a 1989 MHC Class II HLA-Dra Sherman e t a L > 1989 β-Actin Kawamoto et a 1 88; Ng et aL ; 1989 Muscle Creatine Kinase Jaynes e f a \ , 1988; Horlick and Benfield, 1989; Johnson βt α/. > 1989 Prealbumin (Transthyretin) Costa et a , 1988 Elastase I Omitz e , α /., 1987 PROMOTER/ENHANCER REFERENCES Metailothionein Karin et a 1987; Culotta and Hamer, 1989 Collagenase Pinkert et a , 1987; Angel et aL , 1987 Albumin Gene Pinkert et a , 1987; Tranche et aL , 1989, 1990 α-Fetoprotein Godbout e a j , 1988; Campere and Tilghman, 1989 t-Globin Bodine and Ley, 1987; Perez-Stable and Constantini, 1990 β-Globin Trudel and Constantini, 1987 e-fos Cohen et aL , 1987 c-HA-ras Triesman, 1986; Deschamps e t aL > 1985 Insulin Edlund et aL, 1985 Neural Cell Adhesion Molecule Hirsh e , σ /.. 1990 (NCAM) C^l-Antitrypain Larimer ^/., 1990 H2B (TH2B) Histone Hwang et aL , 1990 Mouse or Type I Collagen Ripe et a , 1989 Glucose-Regulated Proteins Chang e/., 1989 (GRP94 and GRP78) Rat Growth Hormone Laxsen e f a ι , 1986 Human Serum Amyloid A (SAA) Edbrooke et aj _, 1989 Troponin I (TN I) Yutzey ^ α /., 1989 Platelet-Derived Growth Factor Pech e , fl /., 1989 Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji er Ω , 1981; Moreau e ^ Ω , 1981; Sleigh and Lockett, 1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbra and Karin, 1986; Kadesch and Berg, 1986; Wang and Calame, 1986; Ondek et α /., 1987; Kuhl e t αL , 1987; Schaffner ^ α /., 1988 Polyoma Swartzendruber and Lehman, 1975; Nasseur et Ω , 1980; Katinka e/ β / , 1980, 1981; Tyndell et α ι, 1981; Dandolo e t α ι, 1983; de Nilliers et αL , 1984; Hen et α l, 1986; Satake et α ι, 1988; Campbell and Nillarreal, 1988 PROMOTER/ENHANCER REFERENCES Retroviruses " Kriegler and Botchan, 1982, 198J; Levinson e t al' ly& ; Kriegler et a h 1983, 1984a, b, 1988; Bosze et a ι, 1986; Miksicek e t ah 1 86; Celander and Haseltine, 1987; Thiesen e t ah 88; Celander et a ι, 1988; Choi et a j , 1988; Reisman and Rotter, 1989 Papilloma Virus Campo et ah 1983; Lusky e f a \ , 1983; Spandidos and Wilkie, 1983; Spalholz e t ah 1985; Lusky and Botchan, 1986; Cripe et a ι, 1987; Gloss e t ah 987; Hirochika et ah 1 87; Stephens and Hentschel, 1987 Hepatitis B Virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau and Lee, 1988; Vannice and Levinson, 1988 Human Immunodeficiency Virus Muesing e t al' 1 87; Hauber and Cullan, 1988; Jakobovits e t al' 1988; Feng and Holland, 1988; Takebe e t ah 1 88; Rosen et a h 1988; Berkhout et ah 1989; Laspia e f a \ , 1989; Sharp and Marciniak, 1989; Braddock et α/., 1989 Cytomegalovirus Weber e f a j , 1984; Boshart e t ah 1985; Foecking and Hofstetter, 1986 Gibbon Ape Leukemia Virus Holbrook e / , 1987; Quinn ^/., 1989 TABLE 2 ΪNDUCffiLE ELEMENTS ELEMENT INDUCER REFERENCES " MTTT Phorbol Ester (T A) Jfalmiter e t ah 1 82; Haslmger Heavy metals and Karin, 1985; Searle ^ α , 1985; Stuart e t ah 1985; Imagawa e t al. > 1987, Karin e t al. , 1987; Angel et a , 1987b; McNeall g f ø/.. 1989 MMTV (mouse mammary Glucocorticoids Huang et ah 1981; Lee et a ι, LEMENT INDUCER REFERENCES tumor virus) 1981; Majors and Varmus, 1983; Chandler et a j , 1983 ; Lee et a ι , 1984; Ponta et a , 1985; Sakai etalΛ^ β-Interferon poly(rI)x Tavernier ^ α /., 1983 poly(rc) Adenovirus 5 E2 Ela Imperiale and Nevins, 1984 Collagenase Phorbol Ester (TPA) Angel et ah 1987a Stromelysin Phorbol Ester (TPA) Angel et ah 1987b SN40 Phorbol Ester (TPA) Angel ef ai, 1987b Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene A23187 Resendez e t al . > 1988 α-2-Macroglobulin IL-6 Kunz ^ Ω /., 1989 Nimentin Serum Riming ^., 1989 MHC Class I Gene H-2 K b Interferon Blanar e ^ ., 1989 HSP70 Ela, SV40 Large T Antigen Taylor et a ι, 1989; Taylor and Kingston, 1990a, b Proliferin Phorbol Ester-TPA Mordacq and Linzer, 1989 Tumor Necrosis Factor FMA Hensel ef a/., 1989 Thyroid Stimulating Thyroid Hormone Chatterjee ^ α /., 1989 Hormone a Gene As used herein, the terms "engineered" and "recombinant" cells are intended to refer to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a cytokine, cytokine receptor or a ribozyme specific for such a polypeptide product, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced exogenous DNA segment. Engineered cells are thus cells having DNA segment introduced through the hand of man. To express a cytokine gene in accordance with the present invention one would prepare an rAAV expression vector that comprises a cytokine-encoding nucleic acid segment under the control of one or more promoters. To bring a sequence "under the control of a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides "downstream" of (ι e _, 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded polypeptide. This is the meaning of "recombinant expression" in this context. Particularly preferred recombinant vector constucts are those that comprise an rAAV vector. Such vectors are described in detail herein. 4.7 PHARMACEUTICAL COMPOSITIONS In certain embodiments, the present invention concerns formulation of one or more of the rAAV compositions disclosed herein in pharmaceutically acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the nucleic acid segment, RNA, DNA or PNA compositions that express a therapeutic gene product as disclosed herein may be administered in combination with other agents as well, such as, e g , proteins or polypeptides or various pharmaceutically-active agents, including one or more systemic or topical administrations of cytokine or cytokine receptor polypeptides. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The rAAV compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may further comprise substituted or derivatized RNA, DNA, or PNA compositions. Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e σ oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation. Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described in U. S. Patent 5,543,158; U. S. Patent 5,641,515 and U. S. Patent 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U. S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office, of Biologies standards. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. 4.8 LIPOSOME-, NANOCAPSULE-, AND MICROPARTICLE-MEDIATED DELΓVERY In certain embodiments, the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV-cytokine constructs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur e t ah 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U. S. Patent 5,741,516, specifically incorporated herein by reference in its entirety). Further, various methods of liposome and liposome like preparations as potential drug carriers have been reviewed (Takakura, 1998; Chandran et α /., 1997; Margalit, 1995; U. S. Patent 5,567,434; U. S. Patent 5,552,157; U. S. Patent 5,565,213; U. S. Patent 5,738,868 and U. S. Patent 5,795,587, each specifically incorporated herein by reference in its entirety). Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen e t al, 1990; Muller e a , 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs (Heath and Martin, 1986; Heath e t ah 1986; Balazsovits e t ah 1989; Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et ah 1987), enzymes (Imaizumi e t ah 1990a; Imaizumi e t ah 1990b), viruses (Faller and Baltimore, 1984), transcription factors and allosteric effectors (Nicolau and Gersonde, 1979) into a variety of cultured cell lines and animals. In addition, several successful clinical trails examining the effectiveness of liposome-mediated drug delivery have been completed (Lopez-Berestein e t al, 1985a; 1985b; Coune, 1988; Sculier e t al, 1988). Furthermore, several studies suggest that the use of liposomes is not associated with autoimmune responses, toxicity or gonadal localization after systemic delivery (Mori and Fukatsu, 1992). Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also tenned multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, f e in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation. In addition to the teachings of Couvreur e t al. (1977; 1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs. In addition to temperature, exposure to proteins can alter the permeability of liposomes. Certain soluble proteins, such as cytochrome c, bind, deform and penetrate the bilayer, thereby causing changes in permeability. Cholesterol inhibits this penetration of proteins, apparently by packing the phospholipids more tightly. It is contemplated that the most useful liposome formations for antibiotic and inhibitor delivery will contain cholesterol. The ability to trap solutes varies between different types of liposomes. For example, MLVs are moderately efficient at trapping solutes, but SUVs are extremely inefficient. SUVs offer the advantage of homogeneity and reproducibility in size distribution, however, and a compromise between size and trapping efficiency is offered by large unilamellar vesicles (LUVs). These are prepared by ether evaporation and are three to four times more efficient at solute entrapment than MLVs. In addition to liposome characteristics, an important determinant in entrapping compounds is the physicochemical properties of the compound itself. Polar compounds are trapped in the aqueous spaces and nonpolar compounds bind to the lipid bilayer of the vesicle. Polar compounds are released through permeation or when the bilayer is broken, but nonpolar compounds remain affiliated with the bilayer unless it is disrupted by temperature or exposure to lipoproteins. Both types show maximum efflux rates at the phase transition temperature. Liposomes interact with cells v α four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time. The fate and disposition of intravenously injected liposomes depend on their physical properties, such as size, fluidity, and surface charge. They may persist in tissues for h or days, depending on their composition, and half lives in the blood range from min to several h. Larger liposomes, such as MLVs and LUVs, are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the capillary endothelium, such as the sinusoids of the liver or spleen. Thus, these organs are the predominate site of uptake. On the other hand, SUVs show a broader tissue distribution but still are -sequestered highly in the liver and spleen. In general, this j n v j vo behavior limits the potential targeting of liposomes to only those organs and tissues accessible to their large size. These include the blood, liver, spleen, bone marrow, and lymphoid organs. Targeting is generally not a limitation in terms of the present invention. However, should specific targeting be desired, methods are available for this to be accomplished. Antibodies may be used to bind to the liposome surface and to direct the antibody and its drug contents to specific antigenic receptors located on a particular cell-type surface. Carbohydrate determinants (glycoprotein or glycolipid cell-surface components that play a role in cell-cell recognition, interaction and adhesion) may also be used as recognition sites as they have potential in directing liposomes to particular cell types. Mostly, it is contemplated that intravenous injection of liposomal preparations would be used, but other routes of administration are also conceivable. Alternatively, the invention provides for pharmaceutically acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland e f a h 1987; Quintanar- Guerrero e f a j., 1998; Douglas e f a h 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded \ n v f v0 . Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention. Such particles may be are easily made, as described (Couvreur e t al, 1980; Couvreur, 1988; zur Muhlen e f af, 1998; Zambaux e t ah 1998; Pinto-Alphandry et a \., 1995 and U. S. Patent 5,145,684, specifically incorporated herein by reference in its entirety). 4.9 ADDITIONAL MODES OF DELIVERY In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the rAAV vector compositions to a target cell or animal. Sonophoresis ( _ e ultrasound) has been used and described in U. S. Patent 5,656,016 (specifically incorporated herein by reference in its entirety) as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U. S. Patent 5,779,708), microchip devices (U. S. Patent 5,797,898), ophthalmic formulations (Bourlais e t ah' 1998), transdermal matrices (U. S. Patent 5,770,219 and U. S. Patent 5,783,208) and feedback-controlled delivery (U. S. Patent 5,697,899), each specifically incorporated herein by reference in its entirety. 4.10 THERAPEUTIC AND DIAGNOSTIC KITS The invention also encompasses one or more compositions together with one or more- pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and/or other components, as may be employed in the formulation of particular rAAV-polynucleotide delivery formulations, and in the preparation of therapeutic agents for administration to a mammal, and in particularly, to a human, for one or more of the cytoldne-deficient conditions described herein. In particular, such kits may comprise one or more rAAV-cytokine composition in combination with instructions for using the viral vector in the treatment of such disorders in a mammal, and may typically further include containers prepared for convenient commercial packaging. As such, preferred animals for administration of the pharmaceutical compositions disclosed herein include mammals, and particularly humans. Other preferred animals include murines, bovines, equines, porcines, canines, and felines. The composition may include partially or significantly purified rAAV-cytokine compositions, either alone, or in combination with one or more additional active ingredients, which may be obtained from natural or recombinant sources, or which may be obtainable naturally or either chemically synthesized, or alternatively produced n v iγ 0 from recombinant host cells expressing DNA segments encoding such additional active ingredients. Therapeutic kits may also be prepared that comprise at least one of the compositions disclosed herein and instructions for using the composition as a therapeutic agent. The container means for such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other container means, into which the disclosed rAAV compositions) may be placed, and preferably suitably aliquoted. Where a second cytokine or cytokine receptor composition is also provided, the kit may also contain a second distinct container means into which this second composition may be placed. Alternatively, the plurality of cytokine or cytokine receptor compositions may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container means. The kits of the present invention will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e .g., injection or blow-molded plastic containers into which the desired vial(s) are retained. 4.11 METHODS OF NUCLEIC ACID DELIVERY AND DNA TRANSFECTION In certain embodiments, it is contemplated that one or more of the rAAV-delivered cytokine-encoding RNA, DNA, PNAs and/or substituted polynucleotide compositions disclosed herein will be used to transfect an appropriate host cell. Technology for introduction of rAAV's comprising one or more PNAs, RNAs, and DNAs into target host cells is well known to those of skill in the art. Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention for use in certain j n v i . 0 embodiments, and under conditions where the use of rAAV-mediated delivery is less desirable. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe e t ah 1 90) DEAE-dextran (Gopal, 1985), electroporation (Wong and Neumann, 1982; Fromm e f a \ , 1985; Tur-Kaspa e t ah 1986; Potter e t ah 1984; Suzuki et al., 1998; Vanbever e f a j., 1998), direct microinjection (Capecchi, 1980; Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley e t ah 1979; Takakura, 1998) and lipofectamine-DNA complexes, cell sonication (Fechheimer e f a h 1987), gene bombardment using high velocity microprojectiles (Yang e f a h 1990; Klein e f a h 1992), and receptor-mediated transfection (Curiel e f a h 1991; Wagner e tah 1992; Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for \ n v j vo or e χ vivo use - 4.12 EXPRESSION IN ANIMAL CELLS The inventors contemplate that a polynucleotide comprising a contiguous nucleic acid sequence that encodes a therapeutic cytokine polypeptide of the present invention may be utilized to treat one or more cellular defects in a transformed host cell. Such cells are preferably animal cells, including mammalian cells such as those obtained from a human or other primate, murine, canine, bovine, equine, epine, or porcine species. In particular, the use of such constructs for the treatment and/or amelioration of eating disorders or neurological dysfunction in a human subject suspected of suffering from such a disorder is highly contemplated. The cells may be transformed with one or more rAAV vectors comprising one or more therapeutic cytokine genes of interest, such that the genetic construct introduced into and expressed in the host cells of the animal is sufficient to alter, reduce, ameliorate or prevent the deleterious or disease conditions either n v if f0 and/or f n v vo . 4.13 TRANSGENIC ANIMALS It is contemplated that in some instances the genome of a transgenic non-human animal of the present invention will have been altered through the stable introduction of one or more of the rAAV-delivered polynucleotide compositions described herein, either native, synthetically modified, or mutated. As used herein, the term "transgenic animal" is intended to refer to an animal that has incorporated exogenous DNA sequences into its genome. In designing a heterologous gene for expression in animals, sequences which interfere with the efficacy of gene expression, such as polyadenylation signals, polymerase II termination sequences, hairpins, consensus splice sites and the like, are eliminated. Current advances in transgenic approaches and techniques have permitted the manipulation of a variety of animal genomes via gene addition, gene deletion, or gene modifications (Franz e f a \., 1997). For example, mosquitos (Fallon, 1996), trout (Ono e t a 1997), zebrafish (Caldovic and Hackett, 1995), pigs (Van Cott e f a \., 1997) and cows (Haskell and Bowen, 1995), are just a few of the many animals being studied by transgenics. The creation of transgenic animals that express human proteins such as α -l-antitrypsin, in sheep (Carver e f a \., 1993); decay accelerating factor, in pigs (Cozzi e t ah 1997), and plasminogen activator, in goats (Ebert e f a f, 1991) has previously been demonstrated. The transgenic synthesis of human hemoglobin (U. S. Patent 5,602,306) and fibrinogen (U. S. Patent 5,639,940) in non-human animals have also been disclosed, each specifically incorporated herein by reference in its entirety. Further, transgenic mice and rat models have recently been described as new directions to study and treat cardiovascular diseases such as hypertension in humans (Franz e t ah 1 97; Pinto-Siestma and Paul, 1997). The construction of a transgenic mouse model has recently been used to assay potential treatments for Alzheimer's disease (U. S. Patent 5,720,936, specifically incorporated herein by reference in its entirety). It is contemplated in the present invention that transgenic animals contribute valuable information as models for studying the effects of cytokine and cytokine receptor compositions on correcting genetic defects and treating a variety of disorders in an animal. 4.14 SELECTION AND CHARACTERIZATION OF CYTOKINE GENETIC CONSTRUCTS The enzyme luciferase is useful as a screenable marker in the context of the present invention (Kang e t al- > 1998). In the presence of the substrate luciferin, cells expressing luciferase emit light that can be detected on photographic or x-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells that are expressing luciferase and manipulate those in real time. The above techniques also could be utilized if the screenable marker is a protein such as green fluorescent protein (gfp). To confirm the presence of the exogenous DNA or "transgene(s)" in the transformed cells, and in particular, a transgene delivered by an rAAV vector composition, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays, such as Southern and Northern blotting, RT-PCRTM and PCRTM; "biochemical" assays, such as detecting the presence of a protein product, e .g., by immunological means (ELISAs and Western blots) or by enzymatic function assay. While Southern blotting and PCRTM may be used to detect the transgene(s) in question, they do not provide information as to whether the gene is being expressed. Expression may be evaluated by RT-PCRTM for mRNA and/or specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression. Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical- chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography. The unique structures of individual proteins offer oppoitunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Transgenic animals are described that synthesize epitope tagged prion proteins as a method of detecting the expressed protein(s) (U. S. Patent 5,789,655, specifically incorporated herein by reference in its entirety). Combinations of approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used. Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and 14 C-acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two. Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the cells of the animal or human. 4.15 DNA INTEGRATION, RNA EXPRESSION AND INHERITANCE Genomic DNA may be isolated from animal cell lines or any animal parts to determine the presence of the exogenously introduced cytokine-encoding genenetic construct through the use of one or more readily-available techniques that are well known to those skilled in the art. The presence of DNA elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCRTM). Using this technique, discreet fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a gene is present in a stable transformant, but does not prove integration of the introduced gene into the host cell genome. In addition, it is not possible using PCRTM techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, j e ., whether transformants are of independent origin. It is contemplated that using PCRTM techniques it would be possible to clone fragments of the host genomic DNA adjacent to an introduced gene. Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, e ., confirm that the introduced gene has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCRTM g „ the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant. It is contemplated that using the techniques of dot or slot blot hybridization which are modifications of Southern hybridization techniques one could obtain the same information that is derived from PCRTM, e „ the presence of a gene. Whereas DNA analysis techniques may be conducted using DNA isolated from any part of an animal, RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues. PCRTM techniques may also be used for detection and quantitation of RNA produced from introduced genes. In this application of PCRTM it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCRTM techniques amplify the DNA. In most instances PCRTM techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridization. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species. 4.16 SELECTABLE MARKERS In certain embodiments of the invention, the delivery of a nucleic acid in a cell, and in particular, an rAAV construct that expresses one or more therapeutic cytokine or cytokine receptor compositions may be identified n γf fQ or j n v f vo by including a marker in the expression construct. The marker would result in an identifiable change to the transfected cell permitting ready identification of expression. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol. Alternatively, enzymes such as herpes simplex virus thymidine kinase (/•&) (eukaryotic) or chlorarnphenicol acetyltransferase (CAT) (prokaryotic) may be employed, as well as markers such as green fluorescent protein, luciferase, and the like. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, as long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art. 4.17 SITE-SPECIFIC MUTAGENESIS Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent polypeptides, through specific mutagenesis of the underlying polynucleotides that encode them. The technique, well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide. In certain embodiments of the present invention, the inventors contemplate the mutagenesis of the contemplated cytokine or cytokine receptor-encoding polynucleotide sequences to alter the activity or effectiveness of such constructs in increasing or altering their therapeutic activity in a transformed host cell. Likewise in certain embodiments, the inventors contemplate the mutagenesis of such genes themselves, or of the rAAV delivery vehicle to facilitate improved regulation of the particular cytokine or cytokine receptor polypeptide 's activity, solubility, stability, expression, or efficacy n y \tγ 0 and/or \ n v / The techniques of site-specific mutagenesis are well known in the art, and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter a specific portion of a DNA molecule. In such embodiments, a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered. As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage. In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as β co \ι polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as β co n cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy e t ah 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis e t ah 1982, each incorporated herein by reference, for that purpose. As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation that result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing. Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent No. 4,237,224, specifically incorporated herein by reference in its entirety. A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCRTM) which is described in detail in U. S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCRTM, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase (e.g., f a q polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated. Preferably reverse transcription and PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art. Another method for amplification is the ligase chain reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308 (specifically incorporated herein by reference in its entirety). In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCRTM, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U. S. Patent No. 4,883,750, incorporated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence. Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880, incorporated herein by reference in its entirety, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected. An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ α -thio]triphosphates in one strand of a restriction site (Walker e t ah 1992), may also be useful in the amplification of nucleic acids in the present invention. Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids that involves multiple rounds of sti'and displacement and synthesis, e nick translation. A similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having a 3' and 5' sequences of non-target DNA and an internal or "middle" sequence of the target protein specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNaseH, and the products of the probe are identified as distinctive products by generating a signal that is released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Thus, CPR involves amplifying a signal generated by hybridization of a probe to a target gene specific expressed nucleic acid. Still other amplification methods described in Great Britain Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR-like, template and enzyme dependent synthesis. The primers may be modified by labeling with a capture moiety e & biotin) and/or a detector moiety e „ enzyme). In the latter application, an excess of labeled probes is added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence. Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh e tal, 989; PCT Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer that has sequences specific to the target sequence. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat-denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target- specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into DNA, and transcribed once again with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target-specific sequences. Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference in its entirety, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of yv co n DNA polymerase I), resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA. PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein by reference in its entirety, disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" (Frohman, 1990), and "one-sided PCR" (Ohara e t al, 989) which are well- known to those of skill in the art. Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu and Dean, 1996, incorporated herein by reference in its entirety), may also be used in the amplification of DNA sequences of the present invention. 4.18 BIOLOGICAL FUNCTIONAL EQUIVALENTS Modification and changes may be made in the structure of the rAAV vector-delivered cytokine compositions, or the polynucleotides and/or encoded polypeptides of the present invention and still obtain a functional molecule that encodes a cytokine or cytokine receptor polypeptide with desirable characteristics. As mentioned above, it is often desirable to introduce one or more mutations into a specific polynucleotide sequence. In certain circumstances, the resulting encoded polypeptide sequence is altered by this mutation, or in other cases, the sequence of the polypeptide is unchanged by one or more mutations in the encoding polynucleotide. When it is desirable to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, second-generation molecule, the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 3. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity. TABLE 3 AMINO ACIDS CODONS Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC uuu Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine He I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gin Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Tip w UGG Tyrosine Tyr Y UAC UAU In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (—1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (—3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, e, still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropatliic indices are within +2 is preferred, those within +1 are particularly preferred, and those within +0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U. S. Patent 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (-0.5); histidine (-0.5); cysteine (—1.0); methionine (-1.3); valine (- 1.5); leucine (—1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within +2 is preferred, those within +1 are particularly preferred, and those within +0.5 are even more particularly preferred. As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. 4.19 RIBOZYMES Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA- protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et a\ , 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech e t «/., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind v ; α specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction. Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech e t ah 1981). For example, U. S. Patent No. 5,354,855 (specifically incorporated herein by reference) reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence- specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon e t ah 1991; Sarver β f ah 1990). Recently, it was reported that ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H- ra>y , c-f os and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme. Six basic varieties of naturally occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds j n trans ( an( l us can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inliibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base- substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf e f ah 1992). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site. The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis § virus, group I intron or RNaseP RNA (in ' association with an RNA guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi e t al. (1992). Examples of hairpin motifs are described by Hampel e t al. (βvcc. Pat. Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et a ι (1990) and U. S. Patent 5,631,359 (specifically incorporated herein by reference). An example of the hepatitis 5 virus motif is described by Perrotta and Been (1992); an example of the RNaseP motif is described by Guerrier-Takada e t al. (1983); Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990; Saville and Collins, 1991; Collins and Olive, 1993); and an example of the Group I intron is described in U. S. Patent 4,987,071 (specifically incorporated herein by reference). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. Thus the ribozyme constructs need not be limited to specific motifs mentioned herein. In certain embodiments, it may be important to produce enzymatic cleaving agents that exhibit a high degree of specificity for the RNA of a desired target, such as one of the cytokine or cytokine receptor sequences disclosed herein. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNA. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required, although in preferred embodiments the ribozymes are expressed from DNA or RNA vectors that are delivered to specific cells. Small enzymatic nucleic acid motifs (e.g., of the hammerhead or the hairpin structure) may also be used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. Alternatively, catalytic RNA molecules can be expressed within cells from eukaryotic promoters (e.g., Scanlon et ah 1991; Kashani-Sabet e t a h 1992; Dropulic e t a i, 1992; Weerasinghe et a 1991; Ojwang e t ah 1992; Chen e tah 1992; Sarver e t ah 1990). Those skilled in the art realize that any ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl. Publ. No. WO 94/02595, both hereby incorporated by reference; Ohkawa β f al' 1992; Taira et aL , 1991; and Ventura et aL , 1993). Ribozymes may be added directly, or can be complexed with cationic lipids, lipid complexes, packaged within liposomes, or otherwise delivered to target cells. The RNA or RNA complexes can be locally administered to relevant tissues e χ vivo' or in vivo through injection, aerosol inhalation, infusion pump or stent, with or without their incorporation in biopolymers. Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595 (each specifically incorporated herein by reference) and synthesized to be tested \ n v if ro and j n v / vo , as described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA targets in other species can be utilized when necessary. Hammerhead or hairpin ribozymes may be individually analyzed by computer folding (Jaeger e t ah 1989) to assess whether the ribozyme sequences fold into the appropriate secondary structure, as described herein. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 or so bases on each arm are able to bind to, or otherwise interact with, the target RNA. Ribozymes of the hammerhead or hairpin motif may be designed to anneal to various sites in the mRNA message, and can be chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman e tal. (1987) and in Scaringe e t al (1990) and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. Average stepwise coupling yields are typically >98%. Hairpin ribozymes may be synthesized in two parts and annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992). Ribozymes may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see e .g, Usman and Cedergren, 1992). Ribozymes may be purified by gel electrophoresis using general methods or by high-pressure liquid chromatography and resuspended in water. Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e .g, Int. Pat. Appl. Publ. No. WO 92/07065; Perreault e t ah 1990; Pieken e t ah 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Patent 5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements. A preferred means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, e f C ) present nearby. Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber e tah 1993; Zhou e t ah 1990). Ribozymes expressed from such promoters can function in mammalian cells (Kashani-Sabet et ah 1992; Ojwang e tah 1992; Chen et ah 1992; Yu e tah 1993; L'Huillier et aL , 1992; Lisziewicz e t ah 1993). Although incorporation of the present ribozyme constructs into adeno-associated viral vectors is preferred, such transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, other viral DNA vectors (such as adenovirus vectors), or viral RNA vectors (such as retroviral, semliki forest virus, sindbis virus vectors). Sullivan e t al ( Int Pa Appl. Publ. No. WO 94/02595) describes general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex v f v0 to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference. Ribozymes of this invention may be used to inhibit gene expression and define the role (essentially) of specified gene products in. the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These studies lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). 5.0 EXAMPLES The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. 5.1 EXAMPLE 1 - LONG-TERM CORRECTION OF OBESITY USING CENTRALLY- DELIVERED RAAV ENCODING ANOREXIGENIC CYTOKINES This example describes the construction of a series of rAAV vectors encoding naturally-occurring anorexigenic cytokines that have been developed for the purpose of long- term body weight (BW and food intake (FI) regulation in obese leptin-resistant patients. Using these vectors it has been shown that rAAV constructs encoding the anorexigenic hormone leptin was efficient in reducing BW and FI in lean as well as in genetically obese animal models after intramuscular m ), intravenous (/ v ) or intracerebroventricular (j cv ) injections. It has also been shown that rAAV encoding cytokines CNTF and leukemia factor (LIF) were efficient in maintaining lower BW in normal rodents for a prolonged period of time. Likewise, it has been shown that rAAV encoding these cytokines rAAV reduced the rate of BW gain up to 15% for 5 months (the duration of the study) after single j cv injection of rAAV in leptin-resistant genetically obese Zuker f a /f a rats. No side or ill effects were encountered during the course of these studies. These data demonstrate that tonic expression of naturally occurring anorexigenic cytokine delivered by rAAV is an efficient mechanism for the regulation of BW and obesity in mammals, and particularly in humans. 5.2 EXAMPLE 2 - SEXUAL DIMORPHISM IN THE RESPONSE TO LEPTIN GENE THERAPY VIΛ AN RAAV VECTOR-BASED DELIVERY SYSTEM This example describes the efficacy of gene therapy with rAAV carrying rat leptin cDNA (rAAV-Leptin) in reducing BW of male and female Sprague Dawley rats. The rAAV construct consisted of rat leptin cDNA driven by a hybrid promoter containing the chicken β- actin promoter and CMV enhancer in a rAAV vector. Adult male and female rats (250-300 g body weight [BW]) were implanted with a permanent injection cannula in the third cerebroventricle. Following post surgical recovery, 8 male and 8 female rats were injected with 5 μl of rAAV-leptin (10 13 particles/ml). Control rats (n=6) each were injected cv with 5 μl of rAAV control virus. BW and 24-hour food intake were monitored weekly. At two weeks post injection there was a significant decrease in BW in female rates vs their initial BS (232.4 ± 5.8 VΛ 250.8 ± 4.6 g. p< 0.05) and also vs controls (264.3 ± 4.5 g, p < 0.010). This decrease was maintained for the 12-week duration of the study. In contrast, whereas control male rats steadily gained BW, rAAV-leptin injected rats maintained their initial BW. A significantly lower BW first apparent at 4 weeks (330 ± 7 g vs 368 ± 11 g, p < 0.05) was maintained for 9 weeks. Interestingly, there was no decrease in the daily food intake or either males or females vs control rats. Serum leptin levels in female rats were significantly reduced in rAAV-leptin treated vs control rats (0.8 ± 0.05 vs 2.3 ± 0.3 ng/ml, p < 0.05). Because leptin was believed to regulate BW in a central fashion, NPY and AGRP gene expression was examined in the hypothalami of female rats by in-situ hybridization. Results showed no differences in the relative expression of these orexigenic signals in rAAV-injected rats vs control rats. In summary, central administration of leptin cDNA in a high titer rAAV vector has a sex-specific effect on the pattern of decrease of BS concomitant with normal food intalce. These studies demonstrate the utility of leptin gene therapy in the treatment of obesity and maintenance of lowered body weight over an extended period of time v { a administration of 5.3 EXAMPLE 3 - AN INTERACTING APPETITE REGULATING NETWORK, BODY WEIGHT REGULATION AND GENE THERAPY An interactive appetite-regulating network (ARN) composed of orexigenic and anorexigenic peptides is the primary neural substrate in hypothalamic control of BW. Peripheral signals ( e g cytokines) acting through NPY ergic signaling in the ARN regulate food intake, energy expenditure and metabolism (Yokosuka e t al. 1999). This example illustrates the utility of rAAV-mediated gene therapy to deliver naturally- occurring anorexigenic cytokines (e.g., leptin and ciliary neurotropic factor, CNTF) in suppressing BW gain in normal and genetically obese animals. The results showed that a single v injection of adeno-associated virus (rAAV) encoding leptin cDNA decreased BW and food intake in leptin-deficient 0 b/ob mice. In out-bred Sprague-Dawley rats, rAAV-leptin delivered ι cv curbed the rate of BW gain and decreased plasma leptin levels without any appreciable effect on food intake for at least 12 weeks. In leptin receptor mutant obese Zucker rats (fa/fa), secretable CNTF in rAAV delivered ; cv significantly reduced the rate of BW gain and decreased average daily food intake over a period of 14 weeks. In summary, these results indicate that the modulation of ARN by anorectic cytokines delivered centrally or peripherally by one or more of the rAAV vectors of the present invention provide a means for controlling BW in a mammal. 5.4 EXAMPLE 4 ~ CONTROL OF BODY WEIGHT IN LEAN AND OBESE RODENT MODELS USING CENTRALLY DELIVERED RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV) VECTORS ENCODING CYTOKINES LEPTIN, CNTF AND LIF This example describes the long-term anorexigenic effect of the hormone leptin and the neurocytokines CNTF and LIF that bind to the IL-6 receptor superfamily. A series of rAAV vectors encoding rat leptin hum LIF and sCNTF (an engineered secreted form) cDNAs were successfully generated. When administered intracerebroventricularly (icv) each of these vectors curtailed BW gain for extended periods of time in normal rats and in genetically obese Zucker (fa/fa) rats - me rAAV-leptin treated lean rats, the reduction in BW resulted from loss of white adipose tissue concomitant with a reduction of blood leptin levels in both males and females; the lean body mass of rAAV-leptin treated animals was essentially indistinguishable from control. Fat mass depletion of rAAV-leptin treated rats as not related to food intake. It apparently resulted from increased energy expenditure through thermogenesis since there was a marked increase in uncoupling protein I (UCPI) mRNA expression in the brown adipose tissue of these animals. To explore the efficacy of putative leptin substitutes for the treatment of genetically obese rodent models of leptin resistance rAAV vectors encoding the cytokines CNTF or LIF cDNAs were administered cv \o fa/fa Zucker rats, and to lean rats. These vectors exerted long-term effects resulting in sustained significant weight loss for up to 20 weeks post injection. The postreceptor signal transduction pathways activated by rAAV- leptin, rAAV-CNTF and rAAV-LIF appeared to be similar. These results demonstrate that gene therapy to centrally deliver cytokines has long-term therapeutic benefits for controlling BW in lean and obese mammals. 5.5 EXAMPLE 5 - ACTIVATION OF STAT-DEPENDENT SIGNALING IN RAT HYPOTHALAMUS BY LEPTIN, CNTF AND LIF TRANSGENES DELIVERED CENTRALLY VIA RAAV VECTORS Studies have shown that related cytokines such an CNTF and LIF that also bind to the IL-6 receptor superfamily, are anorexigenic and share common intracellular signaling pathways. To investigate whether this commonality of biological effects can be gainfully employed to regular body weight (BW), the effects of centrally administered rAAV vectors encoding leptin, CNTF and LIF cDNAs were examined. This example describes the results from a series of studies that showed a single injection of rAAV encoding these cytokines resulted in a significant weight loss for extended periods of time. To study the intracellular mechanism, the early steps of postreceptor signaling activated in the hypothalamus were characterized after a intracerebroventricular (icv) injection of (1) recombinant leptin, CNTF or LIF peptides, and (2) recombinant rAAV vectors encoding the cognate genes. The peptides induced fast (15 to 45 min) activation of STAT-3 by phosphorylation of Tyr705. On the other hand, CNTF and LIF, but not leptin also induced STAT-I phosphorylation on Tyr701. None of these cytokines induced STAT-3 phosphorylation on Ser727. The postreceptor phosphorylation of STAT-3 was also markedly enhanced after administration of rAAV vectors at the time of first indication of significant BW loss. Thus, leptin and the related cytokines CNTF and LIF phosphorylate STAT-3 acutely after an icv injection and this activation is apparently sustained over extended time periods as seen after centrally administered rAAV vectors encoding these cytokines. 5.6 EXAMPLE 6 -- LEPTIN GENE THERAPY REDUCES FOOD INTAKE AND BODY WEIGHT WITH AFFECTING ONSET OF PUBERTY AND REPRODUCTIVE CYCLES The example describes an rAAV genetic construct encoding leptin (rAAV-Ob) that was injected intracerebroventricularly (6 x 10 10 particles in 3 μl) into 24 day-old female Sprague-Dawley rats. The control groups consisted of (a) rats injected icv with rAAV-UF5 (control vector) and (b) unoperated, untreated rats. Rats were maintained on ad-libitum rat chow and water. Food intake and body weight were monitored for 6 months. The results showed that during the pubertal period through day 44, rAAV-lep-bOb-treated rats consumed 26.9% less and 22.8% less during the post-pubertal period through day 180. Body weight gain was accordingly reduced in these rats, (rAAV-Ob: 114.3 + 9.2 g; rAAV-UF5: 206.0 + 4.1 g). Despite this marked suppression of caloric intake and weight gain, the age at vaginal opening of rAAV-Ob-treated rats was not different from that of control rats (rAAV-Ob: 39.9 + 0.6 days; rAAV-UF5: 39.0 + 0.9 days; untreated control: 38.6 + 0.8 days). To examine the effects on estrous cycles, vaginal smears were recorded between days 54-70. All groups exhibited a similar number of estrous cycles of 4-5 day duration (rAAV-Ob: 3.1 + 0.2; rAAN-UF5: 2.9 + 0.2; untreated controls: 2.4 + 0.2). These results show that leptin gene therapy administered prepubertally suppressed food intake and weight gain without altering either the onset of puberty or reproductive cycles in adult life. Consequently, centrally administered leptin gene therapy is a viable therapy to diminish appetite and maintain a lean phenotype for long periods without disrupting the neuroendocrine control of puberty and reproductive cycles. 5.7 EXAMPLE 7 — LONG-TERM SUPPRESSION OF WEIGHT GAIN AND OBESITY BY RAAV-LIF THERAPY Leptin normally regulates body weight by feedback actions on the hypothalamic network that regulates appetite and energy expenditure. However, development of age-related leptin resistance disrupts the intricate balance thereby leading to excessive weight gain and obesity, a major health problem in the United States. A similar age-related pattern of steady weight gain in association with leptin resistance is observed in laboratory rats. This example demonstrates that by circumventing leptin resistance v f a delivery of genes encoding naturally occurring anorectic and weight reduced peptides directly into the hypothalamus, it is possible to ameliorate the age-related weight gain and obesity. Leukemia inhibitor factor (LIF), a potent anorexigenic cytokine, is present in the hypothalamus and LIF receptor mRNA is expressed in hypothalamic sites previously implicated in weight regulation. rAAV vectors encoding human LIF injected once intracerebroventricularly (icv, 10 10 particles in 5 μl) into the adult wild-type female rats completely blocked the weight gain in short-term (6-week) and long-term (20-week) studies without affecting caloric consumption. In fact, weight was maintained at pre-injection range for the duration of the study. This suppression of weight gain resulted from blockage of fat deposition as indicated by significantly reduced blood leptin and insulin concentrations. Further, administration of rAAV-LIF twice (10 10 particles/injections), 2 days apart, was more effective because there was a sigmficant reduction in daily caloric intake along with weight loss and drastic reduction in adiposity, as reflected by low circulating leptin and insulin levels in these rats. Therefore, it has been shown that LIF acts as a novel hypothalamic anorexigenic signal that can be delivered locally into the hypothalamus of adult rats via rAAV vectors to suppress the age-related weight gain and obesity for extended periods, and that the two underlying crucial physiological processes in weight regulation, caloric consumption and energy expenditure, can be differentially manipulated in a dose-dependent manner; low rAAV-LIF titres selectively block weight gain but high rAAV-LIF titres suppress both weight gain and daily caloric consumption. Consequently, central delivery of rAAV-LIF is an efficient therapy to overcome age-related adverse consequences of leptin-resistance on weight and adiposity. 5.8 EXAMPLE 8 - RAAV-CNTF THERAPY RESCUES RETINITIS PIGMINTOSA This example describes the ability of CNTF gene therapy to rescue or delay PR loss in the rds +/- P216L transgenic mouse. Retinas in this animal degenerate due to a combination of a dominant P216L rds transgene and rds +/- haploinsufficiency, thus mimicking rds-caused human RP. An improved version of human CNTF (DH-CNTF) containing 166D and 167H mutations known to enhance its affinity for CNTFRα, was created, thus increasing human CNTF's stability and potency. Four rAAV vectors were tested, two with the CMV promoter (secreted sDH-CNTF and nonsecreted DH-CNTF, defined by the presence or lack of a human growth hormone secretory signal) and two (DH- CNTF and sDh-CNTF) with the mops500 rod opsin promoter (MOPS) that also supports expression efficiently and specifically in rodent rods. The CMV promoter supports expression well in RPE cells and moderately in PR's when injected into the subretinal space. Rescue was initially identified by retinal morphology, expressed as ONL thickness along a full vertical meridian through the optic nerve head (ONH) at P90, 75 days post injection. The results clearly demonstrate that reproducible photoreceptor survival occurred only when the secretable form of CNTF (sDH-CNTF) was expressed from the CMV promoter (p<0.000004). More limited data on 4 animals shows that the CMV-sDH-CNTF rescue effect persists for at least 4 months after injection (p<0.0025). Since both CMV and MOPS promoters drive expression in rods, but only CMV promoter supports RPE expression, rod expression of CNTF, whether secretable or not, is ineffective. This might suggest that rods do not contain the necessary CNTFRα receptor necessary for efficient CNTF interaction. Alternatively (but not excluding the first possibility), rod survival mediated by CNTF may require a CNTF-induced RPE trophic factor. Aspects of both scenarios are being tested. 5.9 EXAMPLE 9 -- INTRATHECAL ADMINISTRATION OF RAAV-CYTOKINE CONSTRUCTS While the rAAV constructs of the present invention are most directly administered v j a central administration, peripheral delivery of the vector constructs may exploit the natural properties of cytokines such as LIF to decrease fat storage in adipocytes locally v f a inhibition of lipoprotein lipase (LPL) activity, decreasing lipogenesis and stimulating lipolysis. This example describes the intrathecal (f ) injection of the rAAV-cytokine constructs directly into cerebrospinal fluid (CSF), to provide an efficient modality of delivery that by-passes the blood-brain barrier and direct transduction of brain tissues. It has been shown that / / -administered rAAV encoding β-glucuronidase (GUS) resulted in a complete elimination of the storage granules in the brain of a mouse model of the mucopolysaccharidosis type VII (MPS VII). Moreover, an ? γ route of administration is relatively benign, and has been routinely used in clinical settings. As such, the inventors contemplate the use of z γ administration routes for delivery of the rAAV-cytokine constructs disclosed herein. Alternatively, the use of traditional modes of delivery, such as those discussed s pra' may be contemplated for administration of particular constructs depending upon the particular therapeutic benefit to be achieved. 5.10 EXAMPLE 10 — CONSTRUCTION OF RAAV VECTORS Several rAAV vectors have been constructed for use in the methods described herein. pTR-sCNTF encodes a secreted form of human CNTF. To construct this vector a synthetic sequence coding for human growth hormone (hGH) signal peptide was fused in-frame to the coding sequence of CNTN cDΝA. pTR-CB A-Ob encodes rat leptin cDΝA under the control of chicken β-actin promoter linked to CMV enhancer (CBA). hLIF-containing constructs encode human LIF cDΝA, cloned and sequenced by a PCR-mediated protocol from LIF- expressing melanoma cell line G-361. In some vectors two transgenes are linked within dicistronic cassettes through an IRES element for coordinate expression. All vectors contain AAV terminal repeats at both sides of the cassette to mediate replication and packaging of the vector. 5.11 EXAMPLE 11 - REGULATION OF BODY WEIGHT BY RAAV-OB, RAAV-SCNTF AND RAAV-HLIF VECTORS It has been recently shown that a single j m injection of rAAV encoding mouse leptin in mice deficient for this hormone ( 0 b/ob) leads to prevention of obesity and diabetes. To design a positive control vector rAAV encoding rat leptin cDNA was constructed (pTR-CBA-Ob). Initially, injection of 10 π particles of this vector has been shown to be more efficient upon f v injection of rAAV-Ob into 0 b/ob mice. The same vector administered cv in lean rats, resulted in a long-term statistically significant reduction of normal BW gain in both males and females. The dramatic reduction of BW resulted from the loss of adipose tissue. In spite of that, the rAAV-Ob-treated animals remained fit and healthy for the duration of the experiment. The protein content of rAAV-Ob-treated rats was essentially indistinguishable from the control. To evaluate CNTF as an anorexigenic agent in the context of rAAV vector rAAV- sCNTF has been tested in lean animals. It was shown that a single j cv injection of 10 10 physical particles of this vector resulted in long-term BW maintenance in lean female rats. Leptin-resistant f a /f a Zucker rats have also shown a statistically significant reduction in BW upon single z - cv injection of this vector. Mutant forms of CNTF (DH-CNTF) have also been constructed that have a higher affinity towards forms of CNTF receptor, as well as CNTF- 1f»ntin • fiι«;inn rrvnctni to Tr> αrlrlitirm it line hppn σt-ιrγuιm tliαt +Vif» > Hτ> inp» T TT? τnpm1-»ι-*r r»-F+l-ι<* showed that hLIF was as potent as leptin in this study. In order to identify cells transduced with rAAV-CBA-hLIF, a GFP reporter gene was placed within the same transgene cassette. In a separate study, the GFP reporter gene linked to the hLIF through an IRES element in one dicistronic transcription unit was also tested. rAAV-CBA-hLIF was injected into spinal cord of a SD rat. One month post-injection the spinal cord was processed, and neurons transduced with rAAV-CBA-hLIF were easily identifiable proving the functional reliability of the GFP reporter gene. The glial cells (oligodendrocytes and astrocytes) have been transduced with rAAV-CBA-l LIF at about 100 times lower rate as compared to neurons. 5.12 EXAMPLE 12 ~ LEPTIN, CNTF AND LIF INITIATE SIMILAR DOWNSTREAM SIGNAL TRANSDUCTION CASCADE IN HYPOTHALAMUS Intraperitoneally injected recombinant CNTF and leptin have been previously shown to activate a similar pattern of STAT factors in hypothalamic satiety centers. On the other hand, it was shown that recombinant leptin administered j v in rats activates STAT-3 phosphorylation, but fails to activate either Jak proteins, or STAT-1 and STAT-5, or even mitogen-activated protein (MAP) kinase in the hypothalamus. Therefore, leptin signaling differs from that of other ligands that bind members of this class of receptor by failing to stimulate measurable responses in any of the known upstream signal transduction proteins. rAAV vectors encoding lipostatic hormone leptin, neurocytokines CNTF and LIF exert strong anorexigenic effect if applied peripherally (rAAV-CB A-Ob), or centrally in genetically obese (rAAV-CB A-OB in 0 b/ob mice and rAAV-CNTF iri fa/fa rats ) > as well as in lean SD rats (all three vectors). Although leptin, CNTF and LIF ligands all interact with different specific receptors, they all activate similar downstream of Jak STAT-3 signaling cascases resulting in loss of BW. Jaks are tyrosine kinases that associate with cytokine and leptin receptors. Upon ligand binding, they activate members of the STAT family through phosphorylation on a single tyrosine. Activated STATs form dimers, translocate to the nucleus, bind to specific response elements in promoters of target genes, and transcriptionally activate these genes. In the case of leptin signaling, the early transduction events preceding STAT-3 phosphorylation, have not been identified yet. To characterize the signaling mechanisms and establish their similarity lean rats were injected cv with recombinant leptin, CNTF and LIF and analyzed the activation of the pathway through STAT-3 signaling in hypothalamus and pituitary. Although the levels of non- phosphorylated-, or serine-phosphorylated STAT-3 remained unchanged, the tyrosine- phosphorylated STAT-3 increased in leptin-injected rats and even more so in CNTF-injected animals. The same observation was also true for pituitary. It is conceivable, though, that the difference in leptin-induced activation pattern of STAT-3 vs those induced with CNTF, or LIF is due to the difference in the up-stream signaling and, as a result, to the timing of STAT-3 phosphorylation. For example, leptin causes tyrosine phosphorylation of STAT-3 in the hypothalamus as early as 5 min. after injection and reaches maximum at 30 min. These data were obtained at 45 min. post administration, by which time the leptin-induced activation might have subsided. Consistent with this assumption are data obtained by Li and Friedman, who isolated an SH2 domain containing protein tyrosine phosphatase 2 (SHP-2) that binds Tyr985 of the leptin receptor. Tyrosyl phosphorylation of SHP-2 j n v jt ro was associated with decreased phosphorylation of Jak-2 but not Ob-Rb or STAT-3. These data suggest that SHP-2 is a component of the leptin signal transduction pathway and it may terminate leptin signaling by decreasing the level of phosphorylation of Jak-2. Likewise, the f cv administration of human recombinant LIF activated the STAT-3 pathway in hypothalamus through the phosphorylation of the tyrosine residue as early as 15 min. post-injection. Similarly to leptin and CNTF, this activation was specific since the serine residue phosphorylation remained unchanged. These data demonstrate that recombinant leptin, CNTF and LIF activate the STAT-3 signal transduction cascade, albeit with a different efficiencies (or timing). 5.13 EXAMPLE 13 - RAAV VECTORS ENCODING TRANSGENES INDUCIBLE WITH DOX This example describes the use of the tet-inducible system in brain using a combination of two rAAV vectors: a) rAAV-tetR-hLIF encoding hLIF under the control of the tet-inducible promoter CMN mir tetO 7 ; b) rAAN-rtTA/tTS encoding transcription transactivator and transcriptional silencer under the control of a strong constitutive CBA promoter. There are several lines of evidence indicating that such an approach will be successful. Recently Haberman e t al (Haberman e t al, 1998) have demonstrated the long-term gene expression in rat brain of a GFP reporter gene under the control of tet-responsive system and the tet- transactivator. j n v j vo studies, carried out for 6 weeks, demonstrated that rAAV vector- mediated expression is sustained until Dox administration upon which reporter gene expression is reduced (Haberman e t al. 1998). However, the tet construct design used by the authors is always characterized by a background leaky transcription from the CMN min tetO 7 promoter, even under conditions of no induction with the effector. On the other hand, delivering a transcription suppressor (silencer) that binds to the tet-responsive promoter in the absence of the effector Dox eliminates the leakiness of transcription. Unfortunately, the packaging size limitation of rAAN does not allow one to fit all four genes (hLIF, GFP, rtTA and tTS) into one vector. Therefore a double vector paradigm that recently was successfully demonstrated by Rendahl e t a i (1998) may be employed for rAAN vectors. Precise control over gene expression is an important issue both in gene therapy protocols and for the exploration of gene function. One of the most elegant regulatable promoter systems in use today, developed by Gossen and co-workers, involves the ]? co jf tetracycline (tet)repressor fused with the C-terminal domain eukaryotic transcriptional activator NP16. This tet-regulatable transcription factor (tTA) specifically ^^-activates artificial "minimal" promoters carrying multiple tet operator sites (tetO ? ). Additionally tet to the system reversibly inhibits binding of tTA to promoter DΝA and blocks gene expression. A variation of the original system involves a tTA derivative that carries four point mutations, (reverse tet- regulatable / rø^-activator, rtTA), which does not ^^.-activate in the default state, but requires a tet-like compound (doxycycline, Dox) for gene activation. However, the drawback of these systems is that tTA and rtTA both display basal leaky expression of the transgene. A recent improvement of the tet system incorporates a tet-controlled /^^-repressor to silence the leaky transcriptional activity of the eukaryotic promoters that are stably integrated into the chromatin of human cells. By fusing the KRAB domain of human Koxl to the £ co u tet repressor, a tet-controlled hybrid protein (TetR-KRAB, or rTS) was generated. The rTS binds to the tetO ? operator sequence in the absence but not in the presence of tet. Therefore, combining rtTA and tTS genes into one system provides mutually exclusive repression/induction of tetO 7 promoter-driven gene. In the absence of Dox complete transcription shut-off is mediated by the tTS bound to the tetO y . Upon adding Dox at concentrations higher than lOng/ml, tTS is displaced by the rtTA trans-activator and transcription is induced. In order to exploit rtTA/tTS system the vector rAAN-rtTA/tTS encoding two chimeric genes rtTA and tTS was constructed. Expression of both genes linked by IRES element into one dicistronic cassette, is mediated by a constitutive CBA promoter. The CBA promoter is active in all cell types tested, including neurons. To test this vector a transient transfection study was performed using HEK292 cells, with a destabilized GFP reporter gene driven by the CMV miπ tetO 7 promoter (pTRE-dEGFP, Clontech). This study clearly demonstrates that the combination of two rAAV transfer vectors, one carrying transgene under the control of CMV mjn tetO 7 promoter, the other a combination of transcription activator and repressor driven by the strong housekeeping promoter, provides tight control over expression using Dox. This Dox-inducible system is very useful for a temporal conditional expression of transgene, whose product exerts a strong biological effect potentially leading to side effects. Therefore an rAAN vector expressing hLIF under control of the inducible CMN min tetO 7 promoter (pTR-tetR-hLIF) may provide particular advantages upon j cv injection in the methods disclosed herein. 5.14 EXAMPLE 14 ~ PRODUCTION OF RAAV VECTORS Traditional protocols to produce rAAV vectors have generally been based on a three- component system. One component of this system is a proviral plasmid encoding the recombinant DNA to be packaged as rAAV. This recombinant DNA is located between 145 base pair (bp) AAV-2 inverted terminal repeats (ITRs) that are the minimal c j s acting AAV-2 sequences that direct replication and packaging of the vector. A second component of the system is a plasmid encoding the AAV-2 genes, re p and ca p. The AAV-2 re p gene encodes four Rep proteins (Rep 78, 68, 52 and 40) that act in trans to replicate the rAAV genome, resolve replicative intermediates, and then package single-stranded rAAV genomes. The AAV-2 ca gene encodes the three structural proteins (VP1, VP2, and VP3) that comprise the virus capsid. Because AAV-2 does not proficiently replicate on its own, the third component of a rAAV packaging system is a set of helper functions from another DNA virus. These helper functions create a cellular environment in which rAAV replication and packaging can efficiently occur. The helper functions provided by adenovirus (Ad) have almost exclusively been used to produce rAAV and are encoded by the genes Ela, El b, E2a, E4orf6, and VA RNA. While the first two components of the system are generally introduced into cells in which replication and packaging is to occur by transfection, ad helper functions are introduced by superinfection with wild type Ad virus. The traditional rAAV production techniques are limited in their ability to produce large quantities of vector because of inherent inefficiencies in transfection. Serious difficulties are also encountered when the scale of transfection is increased. The requirement for wild type Ad may also reduce the amount of rAAV produced since Ad may compete for cellular and viral substrates that are required for viral replication but are present only in limiting amounts. Another problem encountered in traditional production protocols is that superinfection with Ad requires development of effective procedures for purification of Ad from the rAAV produced. While these purification processes are generally successful at eliminating Ad contamination of rAAV preparations, they also reduce rAAV titers. Stringent assays for Ad contamination of rAAV are also necessary. To produce rAAV, a double co-transfection procedure is used to introduce a rAAV transfer vector plasmid together with pDG (Grimm e t al. 1998) AAV helper plasmid carrying the AAV rep and cap genes, as well as Ad helper genes required for rAAV replication and packaging at a 1 :1 molar ratio. Plasmid DNA used in the transfection is purified by a conventional alkaline lysis/CsCl gradient protocol. The transfection is carried out as follows: 293 cells are split 1:2 the day prior to the experiment, so that, when transfected, the cell confluence is about 75-80%. Ten 15-cm plates are transfected as one batch. To make CaPO 4 precipitate 0.7 mg of pDG are mixed with 180 μg of rAAV transfer vector plasmid in a total volume of 12.5 ml of 0.25 M CaCL.. The old media is removed from the cells and the formation of the CaPO 4 -precipitate is initiated by adding 12/5 ml of 2xHBS pH 7.05 (pre- warmed at 37°C) to the DNA-CaCl 2 solution. The DNA is incubated for 1 min; and transferring the mixture into pre-warmed 200 ml of DMEM- 10% FBS then stops the formation of the precipitate. Twenty two ml of the media is immediately dispensed into each plate and cells are incubated at 37°C for 48 firs. The CaPO 4 -precipitate is allowed to stay on the cells during the whole incubation period without compromising cell viability. Forty-eight hr post- transfection cells are harvested by centrifugation at 1,140 x g for 10 min. Cells are then lysed in 15 ml of 0.15 m MaCl, 50 mM tris HCI pH 8.5 by 3 freeze/thaw cycles in dry ice-ethanol and 37°C baths. Benzonase (Ny corned Pharma A/S, pure grade) is added to the mixture (50 U/ml, final concentration) and the lysate is incubated for 30 min at 37°C. The lysate is clarified by centrifugation at 3,700 g for 20 min and the virus-containing supernatant is further purified using a discontinuous density gradient. The typical discontinuous step gradient is formed by underlayering and displacing the less dense cell lysate with Iodixanol, 5,5"[(2-hydroxi-l-3-propanediyl)-bis(acetylamino)] bis [N,N'bi ,(2,3dihydroxypropyl-2-4,6-triiodo-l,3-enzenecarboxamide], prepared using a 60% (wt./vol.) sterile solution of OptiPrep (Nycomed). Specifically, 15 ml of the clarified lysate are transferred into Quick-Seal Ultra-Clear 25 x 89 mm centrifuge tube (Beckman) using a syringe equipped with 1/27 x 89 mm spinal needle. Care is taken to avoid bubbles, which would interfere with subsequent filling and sealing of the tube. A variable speed peristaltic pump, Model EP-1 (Bio-Rad), is used to underlay in order: 9 ml of 15% iodixanol and 1 M NaCl in PBS-MK buffer containing Phenol Red (2.5 μl of a 0.5% stock solution per ml of the iodixanol solution); 5 ml of 40% iodixanol in PBS-MK buffer; and finally, 5 ml of 60% iodixanol in PBS-MK buffer containing Phenol Red (0.1 μl 1). Tubes are sealed and centrifuged in a Type 70 Ti rotor (Beckman) at 350,000 x g for 1 hr at 18°C. Four ml of the clear 40% step is aspirated after puncturing the tube on the side with a syringe equipped with an 18-gauge needle with the bevel uppermost. The iodixanol fraction is further purified using conventional Heparin agarose affinity chromatography. For chromatography, typically, a pre-packed 2.5 ml Heparin agarose Type I column (Sigma) is equilibrated with 20 ml of PBS-MK under gravity. The rAAV iodixanol fraction is then applied to the pre-equilibrated column, and the column is washed with 10 ml of PBS-MK. rAAV is eluted with the same buffer containing 1M NaCl. After applying the elution buffer, the first 2 ml of the eluant are discarded, and the virus is collected in the subsequent 3.5 ml of elution buffer. Virus is then concentrated and desalted by centrifugation through the BIOMAX 100 K filter (Millipore) according to the manufacturer instructions. The high salt buffer is changed by repeatedly diluting the concentrated virus with the Lactated Ringer's solution and repeating the centrifugation. To characterize the quality of the virus, two assays are used to titer both physical and infectious rAAV particles. A conventional dot-blot assay or quantitative competitive PCRTM (QR PCRTM) assay are used to determine physical particle titers. Infectious titers are determined by infectious center assay (ICA) and fluorescent cell assay (FCA), which scores for expression of GFP. QC PCRTM method is based on competitive co-amplified of a specific target sequence with internal standard plasmid of known concentration in on reaction tube. It provides precise and fast quantitation of viral particles. The internal standard must hare primer recognition sites with the specific template. Both the specific template and the internal standard must be PCRτM-amplified with the same efficiency and it must be possible to analyze the PCRTM- amplified products separately. The easiest way to distinguish between the template and the internal standard is to incorporate a size difference in the two products. This can be achieved, for example, by constructing standards having the same sequence as the specific target but containing a deletion. Quantitation is then performed by comparing the PCRTM signal of the specific template with the PCRTM signal obtained with known concentrations of the competitor (the internal standard). The purified viral stock is first treated with DNAsel to digest any contaminating unpackaged DNA. Ten μl of a purified virus stock is incubated with 10 U of DNA se I (Boehringer) in a 100 μl reaction mixture, containing 50 mM Tris HCI, pH 7.5, 10 mM MgCl 2 for 1 hr at 37°C. At the end of the reaction, 10 μ l of 10X Protinase K buffer (10 mM Tris HCI, pH 8.0, 10 mM EDTA, 1% SDS final concentration) is added, followed by the addition of 1 μ l of Proteinase K (18.6 mg/ml, Boehringer). The mixture is incubated at 37°C for one hour. Viral DNA is purified by phenol/chloroform extraction (twice), followed by chloroform extraction and ethanol precipitation using 10 μg of glycogen as a carrier. The DNA pellet is dissolved in 100 μl of water. QC PCRTM reaction mixtures each contains 1 μl of the diluted viral DNA and two-fold serial dilutions of the internal standard plasmid DNA, such as pdl- GFP. The most reliable range of standard DNA was found to be between 1 and 100 pg. And aliquot of each reaction is the analyzed by 2%> agarose gel electrophoresis, until two PCRTM products are resolved. The analog image of the ethidium bromide stained gel is digitized using and ImageStore 7500 system (UVP). The densities of the target and competitor bands in each lane are measured using the ZERO-Dscan Image Analysis System, version 1.0 (Scanalytics) and their ratios are plotted as a function of the standard DNA concentration. A ration of 1, at which the number of viral DNA molecules equals the number of competitor DNA molecules is used to determine the DNA concentration of the virus stock. In case of rAAV-CB A-Ob, as well as the rAAV-rtTA/tTS vector, for which no plasmid DNA carrying a deleted standard template is available yet, the titer of physical rAAV particles is determined using conventional dot-blot assay and viral DNA purified as described above. Sample of serially diluted respective plasmid of sequence is used as a hybridization probe. A modification of the previously published protocol (McLaughlin e t al, 1988) is used to measure the ability of the virus to infect C12 cells, unpackage, and replicate. Briefly, C2 cells containing integrated wtAAV rep and cap genes (Clark e t al, 1995), are plated in a 96- well dish at about 75% confluence and infected with Ad5 at the M.O.I of 20. One μl of serially diluted rAAV-sCNTF is visually scored using a fluorescence microscope. High sensitivity CHROMA filter #41012 High Q FITC LP is used to monitor the fluorescence. To calculate the titer by hybridization, cells are harvested and processed essentially ad described earlier (McLaughlin e t al, 1988). 5.15 EXAMPLE 15 - RHSV-1 VECTORS FOR THE PRODUCTION OF RAAV In order to make an rAAV production system which did not possess the restrictions to large-scale rAAV production inherent in traditional Ad-based transfection systems, the use of another DNA helper virus of AAV, herpes simplex virus type I (HSV-1), was investigated for the production of rAAV. Like Ad, HSV-1 is able to fully support AAV replication and packaging. While little is known about the nature of the interactions between HSV01 and AAV, the minimal set of HSV-1 genes required to replicate and package AAV has been determined to be UL5, UL8, UL52 and UL29. These genes encode components of the HSV-1 core replication machinery and by themselves are able to form nuclear prereplication centers that develop into mature replication foci. The goal was to create a single infectious agent capable of delivering all AAV-2 genes and HSV-1 helper virus functions required to produce rAAV. In this recombinant HSV-1, the AAV-2 re p and ca p genes are recombined into the viral genome (vv thin the tk gene) to create the recombinant vector d27.1-rc- Therefore, use of this infectious agent along with a rAAV proviral cell line would eliminate restrictions to large- scale rAAV production inherent in transfection protocols. In addition, use of a mutant HSV-1 that was replication defective and not complemented by the rAAV proviral cell line would eliminate the HSV-1 helper virus from the rAAV preparation. Thus, the two significant problems associated with Ad based systems would not be encountered. The recombinant virus expressing re p and ca p, d27.1-rc was constructed by homologous recombination techniques. The HSV-1 background used to make the recombinant vector expressing the AAV-2 re p and ca p genes was d27.1- This mutant vector, d27.L does not produce ICP17. This vector was chosen because it is replication defective and should be less cytotoxic in a non-permissive cell line that wt HSV-1. The virus d27.1 sm l does, however, express the early genes known to be required for rAAV production and thus should still provide efficient helper function for rAAV production. Using an ICP27 mutant as the helper virus for rAAV production has additional advantages. Host cell splicing of messenger RNA is inhibited by ICP27. In addition, d27.1 over-expresses for AAV replication and packaging. 5.16 EXAMPLE 16 ~ A RECOMBINANT HSV-1 EXPRESSING AAV-2 j ^p AND CAP SUPPORTS EFFICIENT RAAV PRODUCTION The ability of the recombinant virus, d27.1-rc to produce rAAV was analyzed. The amount of AAV-GFP produced after 293 cells are transfected with a proviral plasmid and superinfected with d27.1-rc nas b een determined. Efficient rescue of rAAV from the plasmid pTR-UF5, a proviral rAAV plasmid that encodes the green fluorescent protein (GFP), is observed. Up to 380 expression unit (e.u.) of AAV-GFP are produced per cell after superinfection with d.27.1-rc at a ^01 often. Recombinant d27.1-rc 1S even more effective at producing rAAV from the rAAV proviral cell line 293:AAV-GFP. At a M.O.I. of ten, 480 e.u. of AAV-GFP per cell can be produced from this cell line. The amount of AAV-GFP produced from the cell line is a function of the M.O.I. of d27.1-rc- Increasing the M.O.I. to ten resulted in an increase in rAAV production. At M.O.I.s higher than ten, significantly less rAAV is produced. The efficient production of rAAV using d27.1-rc nas b een maintained when the scale of infection was increased 100-fold in tissue culture flasks. When 10 9 293: AAV-GFP cells are infected with d27.1-rc' over 350 e.u. of AAV-GFP are produced per cell. Importantly, rcAAV has not been observed in any large preparation of AAV-GFP made using d27.1-rc (*)• 5.17 EXAMPLE 17 ~ CYTOKINE AND CYTOKINE RECEPTOR POLYPEPTIDE-ENCODING NUCLEIC ACID SEGMENTS The inventors contemplate the use of one or more of the cytokine and cytokine receptor DNA sequences illustrated in Table 4 in the preparation of rAAV vector-based constructs in the practice of the present invention. In illustrative embodiments, the inventors contemplate the use of mammalian cytokine and cytokine-receptor encoding polynucleotides, and in particular, the use of human cytokine and cytokine-receptor encoding polynucleotides in creation of the rAAV vector constructs for therapeutic administration. TABLE 4 POLYNUCLEOTIDE SEQUENCES ENCODING CYTOKINES AND CYTOKINE RECEPTORS USEFUL IN THE PRACTICE OF THE PRESENT METHODS MOLECULE GENBANK ACCESSION NUMBER Rat Leptin D49653 Mouse Leptin Y 10297 Human Leptin NM000230 Rat Leptin Receptor D84126; U52966 Mouse Leptin Receptor Y 10298 Pig Leptin Receptor AJ223163 ; AF092422 Horse Leptin Receptor AF139663 Sheep Leptin Receptor U62124 Human Leptin Receptor NM002303; AH003667; U59261; U59262; U59263 Pig CNTF U57644 Human CNTF NM000614 Rat CNTF receptor S54212 Human CNTF receptor M73238; NM001842 Human Leukemia Inhibitory Factor NM002309 Pig LIF Receptor U97364 Rat LIF Receptor α D86345 Mouse LIF Receptor X99779; X99778 Human LIF Receptor NM002310; X61615 Human Interleukin-1 (IL-1) E00909; E00619 Human IL-1 Receptor, type I NM000877; NM003856 Human IL-1 Receptor, type II NM004633 Human IL-1 Receptor-like 2 (IL1RL2) NM003854 Human Interleukin-2 (IL-2) E00573; S77834; S77835 Human IL-2 like E02011; E00211 Human IL-2 Receptor X01057; E00727 Human IL-2 Receptor α (IL2RA) NM000417 Human IL-2 Receptor β (IL2RB) NM000878 Human IL-2 Receptor γ (IL2RG) NM000206; LI 9546 Human Interleukin-6 (IL-6) S56892 Human IL-6 Receptor (IL6R) NM000565 Human Interleukin- 10 (IL- 10) U16720; AF043333 Human IL-10 Receptor α (ILIORA) NM001558 Human IL-10 Receptor β (IL10RB) NM000628 Brain-Derived Neurotrophic Factor (BDNF) M61176; NM001709 6.0 REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 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While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims: CLAIMS: An adeno-associated viral vector comprising at least a first nucleic acid segment encoding a cytokine or a cytokine receptor polypeptide operably linked to at least a first promoter capable of expressing said segment in a host cell transformed with said vector. The vector of claim 1 , wherein said nucleic acid segment encodes a cytokine. The vector of claim 1 or claim 2, wherein said nucleic acid segment encodes a cytokine selected from the group consisting of an interleukin polypeptide, leptin, leukemia inhibitory factor (LIF), and a neurotrophic factor polypeptide. The vector of any preceding claim, wherein said nucleic acid segment encodes a neurotrophic factor polypeptide selected from the group consisting of brain-derived neurotrophic factor (BDNF) polypeptide and ciliary neurotrophic factor (CNTF) polypeptide. The vector of any preceding claim, wherein said nucleic acid segment encodes an interleukin polypeptide selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, and IL-13. The vector of any preceding claim, wherein said nucleic acid segment encodes an interleukin polypeptide selected from the group consisting of human IL-1 type I, IL- 1, type II, IL-2, IL-2RA, IL-2RB, IL-2RG, IL-6, IL-6R, IL-10, IL-10RA, and IL- lORB. The vector of any preceding claim, wherein said nucleic acid segment encodes a cytokine receptor polypeptide selected from the group consisting of an orexigenic receptor polypeptide, an interleukin receptor polypeptide and a neurotrophic factor receptor polypeptide. The vector of any preceding claim, wherein said nucleic acid segment encodes a cytokine receptor polypeptide selected from the group consisting of a leptin receptor polypeptide, a LIF receptor polypeptide, a BDNF receptor polypeptide, and a CNTF receptor polypeptide. The vector of any preceding claim, wherein said nucleic acid segment encodes a polypeptide of human, murine, porcine, bovine, equine, epine, caprine, or lupine origin. The vector of any preceding claim, wherein said nucleic acid segment encodes a polypeptide that comprises an amino acid sequence selected from any one of the GenBank accession sequences identified in Table 4. The vector of any preceding claim, comprised within a recombinant adeno- associated viras virion. The vector of any preceding claim, comprised within a host cell. The vector of any preceding claim, comprised within a mammalian host cell. The vector of any preceding claim, comprised within a human host cell. The vector of any preceding claim, comprised within a human. A plurality of rAAV virions comprising the vector of any preceding claim. A host cell comprising the vector of any one of claims 1 to 15 or the plurality of virions of claim 16. A composition comprising the vector of any one of claims 1 to 15, or the plurality of rAAV virions of claim 16, or the host cell of claim 17. The composition of claim 18, further comprising a pharmaceutical excipient. The composition of claim 18 or claim 19, further comprising a nanoparticle, a nanocapsule, a liposome, a lipid, or a lipid complex. Use of a vector in accordance with any one of claims 1 to 15, or a composition in accordance with claim 18 or claim 19, in the manufacture of a medicament. Use of a vector in accordance with any one of claims 1 to 15, or a composition in accordance with claim 18 or claim 19, in the manufacture of a medicament for treating a cytokine or cytokine receptor polypeptide deficiency condition in a mammal. Use according to claim 22, wherein said deficiency condition results in an eating disorder or an increased body weight in said mammal. Use according to claim 22, , wherein said deficiency condition results in an eating disorder selected from the group consisting of obesity, anorexia, overeating, excessive body weight gain, and bingeing. A method for treating or ameliorating the symptoms of a cytokine or cytokine receptor polypeptide deficiency condition in a mammal, said method comprising administering to said mammal a vector according to any one of claims 1 to 15, a plurality of rAAV virions according to claim 16, or a composition according to any one of claims 18 to 20, in an amount and for a time sufficient to treat or ameliorate the symptoms of said deficiency in said mammal. The method claim 25, wherein said deficiency condition results in an eating disorder or an increased body weight in said mammal. The method of claim 25 or claim 26, wherein said deficiency condition results in an eating disorder selected from the group consisting of obesity, anorexia, overeating, excessive body weight gain, and bingeing. The method of any one of claims 25 to 27, wherein said vector, said plurality of rAAV virions, or said composition is administered to said animal in an amount and for a time sufficient to decrease the body weight of said mammal, or to decrease the rate of body weight gain in said mammal. The method of any one of claims 25 to 28, wherein said vector, said plurality of rAAV virions, or said composition is administered to said animal intramuscularly or intrathecally. A kit for treating or ameliorating the symptoms of a cytokine or cytokine receptor polypeptide deficiency condition in a mammal comprising: (i) a vector according to any one of claims 1 to 15, a plurality of rAAV virions according to claim 16, or a composition according to any one of claims 18 to 20; and (ii) instructions for using said kit. A kit for treating or ameliorating the symptoms of an eating disorder in a mammal comprising (i) a vector according to any one of claims 1 to 15, a plurality of rAAV virions according to claim 16, or a composition according to any one of claims 18 to 20; and (ii) instructions for using said kit.

Inventors: HAUSWIRTH WILLIAM W, KALRA PUSHPA S, KALRA SATYA P, MOLDAWER LYLE L, MUZYCZKA NICHOLAS, ZOLOTUKHIN SERGEI, HAUSWIRTH, WILLIAM, W., KALRA, PUSHPA, S., KALRA, SATYA, P., MOLDAWER, LYLE, L., MUZYCZKA, NICHOLAS, ZOLOTUKHIN, SERGEI
Applicants: HAUSWIRTH WILLIAM W, KALRA PUSHPA S, KALRA SATYA P, MOLDAWER LYLE L, MUZYCZKA NICHOLAS, UNIV FLORIDA, ZOLOTUKHIN SERGEI, HAUSWIRTH, WILLIAM, W., KALRA, PUSHPA, S., KALRA, SATYA, P., MOLDAWER, LYLE, L., MUZYCZKA, NICHOLAS, UNIVERSITY OF FLORIDA, ZOLOTUKHIN, SERGEI
Application year: 2001
Main classification: C12N15
IPC codes: A61K48, A61P3, C07K14