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Title:  Use of insulin-like growth factor-I in muscle

United States Patent:  6,723,707

Issued:  April 20, 2004

Inventors:  Sweeney; H. Lee (Philadelphia, PA); Rosenthal; Nadia A. (Concord, MA)

Assignee:  The Trustees of the University of Pennsylvania (Philadelphia, PA); Massachusetts General Hospital (Charlestown, MA)

Appl. No.:  510268

Filed:  February 22, 2000

Abstract

The invention relates compositions and method for the use of insulin-like growth factor to enhance muscle mass and strength.

SUMMARY OF THE INVENTION

The invention relates to a method of increasing vertebrate muscle mass and muscle strength. The method comprises administering a muscle enhancing dose of an isolated nucleic acid encoding Insulin-like Growth Factor I (IGF-I), or a modification or biologically active portion thereof, intramuscularly into a vertebrate, wherein the isolated nucleic acid is expressed in muscle cells, thereby increasing the muscle mass and the muscle strength in the muscle of the vertebrate.

In one aspect, the vertebrate is selected from a group consisting of rat, mouse, cat, dog, horse, cow, pig, sheep, goat, fish, bird, and human.

In a preferred embodiment, the vertebrate is a human.

In another preferred embodiment, the IGF-I is of the same species as the vertebrate.

In another aspect, the isolated nucleic acid is contained within a virus vector.

In yet another aspect, the muscle enhancing dose ranges from between about 1010 to about 1012 recombinant virus vector particles per gram of muscle.

In yet another aspect; the method further comprises administering to the vertebrate fibroblast growth factor or neurotropin.

The invention also relates to an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein the IGF-I coding region is flanked on the 5' side by an SV40 intron sequence and wherein the IGF-I coding region is flanked on the 3' end by an SV40 polyadenylation signal sequence.

In one aspect, the muscle specific promoter/regulatory region is selected from a group consisting of the myosin light chain 1/3 promoter/enhancer, the skeletal .alpha.-actin promoter, the muscle creatine kinase promoter/enhancer and a muscle specific troponin promoter.

In a preferred embodiment, the muscle specific troponin promoter is the fast troponin C promoter/enhancer.

In another preferred embodiment, the muscle specific promoter/regulatory region is the myosin light chain 1/3 promoter/enhancer.

In another aspect, the muscle specific promoter/regulatory region further comprises an enhancer element operably linked to the IGF-I coding region.

In a preferred embodiment, the enhancer is the myosin light chain 1/3 enhancer.

Also included in the invention is a composition comprising a recombinant virus vector comprising an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5' side by an SV40 intron sequence and wherein said IGF-I coding region is flanked on the 3' end by an SV40 polyadenylation signal sequence. The muscle specific promoter/regulatory region further comprises an enhancer element operably linked to the IGF-I coding region and the enhancer is the myosin light chain 1/3 enhancer.

In one aspect, the recombinant virus vector is selected from the group consisting of an adeno-associated virus, an adenovirus and a herpes simplex virus.

In a preferred embodiment, the recombinant virus vector is an adeno-associated virus.

Also included in the invention is a cell comprising an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5' side by an SV40intron sequence and wherein said IGF-I coding region is flanked on the 3' end by an SV40 polyadenylation signal sequence.

The invention further includes a cell comprising a recombinant virus vector comprising an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5' side by an SV40 intron sequence and wherein said IGF-I coding region is flanked on the 3' end by an SV40 polyadenylation signal sequence. The muscle specific promoter/regulatory region further comprises an enhancer element operably linked to the IGF-I coding region and the enhancer is the myosin light chain 1/3 enhancer.

The invention additionally includes a kit for increasing muscle mass and muscle strength in a vertebrate. The kit comprises a muscle enhancing dose of an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5' side by an SV40 intron sequence and wherein said IGF-I coding region is flanked on the 3' end by an SV40 polyadenylation signal sequence, wherein the isolated nucleic acid is expressed in vertebrate muscle cells, and wherein the kit further comprises an applicator for delivering the muscle enhancing dose, and instructions for the use of the kit.

Also included in the invention is a non-human transgenic vertebrate animal comprising an isolated nucleic acid encoding IGF-I, or a modification or biologically active portion thereof.

In one aspect, the IGF-I is operably linked to a muscle specific promoter/regulatory sequence at the 5' end of the IGF-I and a polyadenylation termination signal at the 3' end of the IGF-I.

In another aspect, the muscle specific promoter/regulatory sequence is selected from the group consisting of the myosin light chain 1/3 promoter/enhancer, the skeletal .alpha.-actin promoter, the muscle creatine kinase promoter/enhancer and a muscle specific troponin promoter.

In yet another aspect, the non-human transgenic vertebrate animal is selected from the group consisting of rat, mouse, cat, dog, horse, cow, pig, sheep, goat, fish, bird, and human.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered in the present invention that introduction of an isolated nucleic acid encoding IGF-I into adult muscle cells, wherein IGF-I is then expressed in the adult muscle cells, results in hypertrophy of the muscle and/or reversal of age-related muscular atrophy. More importantly, in contrast to prior art procedures, the overall strength of the muscle is enhanced. Without wishing to be bound by theory, it may be that recombinant virus-mediated muscle fiber expression of IGF-I activates satellite cells where prior art methods failed to do so in that prior art procedures did not sufficiently elevate local levels of IGF-I in the muscle. Alternatively, it may be that systemic IGF-I failed to gain access into and to activate satellite cells. In any event, the present invention has circumvented the obstacles which prior art compositions and procedures failed to overcome.

Senescence in mammals is accompanied by loss of strength and endurance in the skeletal musculature. This is due, inter alia, to a diminution in the average diameter of muscle fibers, a shift to less powerful muscle fiber types, decreased mitochondrial volume, and decreased mitochondrial efficiency. The most significant loss of muscle mass in adult muscle is due to the reduction in the number and/or size of the largest, most powerful muscle fibers, the type IIb and type IIx fibers.

The invention relates to the introduction of IGF-I into adult muscle. In a preferred embodiment, a recombinant viral vector delivery system, i.e., an adeno-associated viral (AAV) vector, was used to introduce nucleic acid encoding IGF-I into the muscle tissue of a mammal. However, the invention should not be construed to be limited to any particular vector or to any particular strain of AAV. Rather, the invention should be construed to include all other means of introducing nucleic acid encoding IGF-I into muscle cells including, but not limited to, any strain of AAV which is capable of delivering genes to muscles cells (for example, including, but not limited to, AAV- 1, AAV-3, AAV-4 and AAV-6), other viruses including adenoviruses, herpes simplex viruses, retroviruses, and the like. The invention also includes plasmid-based delivery vectors as well as the administration of DNA in the absence of any vector such as administration of naked DNA by a "gene gun" approach, or the administration of coated DNA, or any other method facilitating DNA entry into muscle tissue.

By the term "naked DNA" as used herein, is meant DNA, which, irrespective of the route or method of administration, is administered to an animal in the absence of any substantial amount of protein, lipid, or any other compound either covalently bound to or non-covalently associated with the DNA. For example, naked DNA includes but is not limited to a plasmid or an isolated nucleic acid fragment encoding a sequence of interest whether naturally or synthetically derived.

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

As used herein, the term "substantially purified" describes a compound, e.g., a protein or polypeptide which has been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

The term "coated DNA," as used herein, describes a DNA associated with a non-proteinaceous substance which may be administered to an animal. Such non-proteinaceous substances include but are not limited to lipids, polylysine, glycosylated polylysine, and the like.

In a preferred embodiment, the IGF-I coding sequence used in the compositions and methods of the invention is the 0.6 kb rat IGF-I cDNA. The invention, however, is not limited solely to this IGF-I DNA. Instead, the invention encompasses any and all IGF-I coding sequences having the biological activity of IGF-I including promoting muscular hypertrophy and enhancing muscle strength as described herein. For example, the invention should be construed to include all mammalian IGF-I sequences which are either known or unknown. The preferred type of IGF-I to be used depends upon the species of animal being treated, in that, the it is preferred that the IGF-I be species matched. Thus, for example, when a human is being treated, the preferred type of IGF-I is human IGF-I, etc. Although all forms of IGF-I are likely to have an effect in different animals, species matching will void potential adverse immunological complications stemming from the induction of an immune response to an IGF-I of a different species in any given animal. Sources of IGF-I from different animal species include the following types of IGF-I and accompanying GenBank Accession Numbers: Cervus elaphus insulin-like growth factor I (IGF4) mRNA, Accession No. U62106; Equus caballus insulin-like growth factor I precursor (IGF-I) mRNA, Accession No. U28070; Goat mRNA for insulin-like growth factor-I, Accession No. D11378; Oryctolagus cuniculus insulin-like growth factor I precursor (IGF-I) mRNA, Accession No. U75390; Pig insulin-like growth factor I (pIGF-I) mRNA, Accession No. M31175; Ovis aries insulin-like growth factor I (IGF-I) mRNA, Accession No. M89787; Human insulin-like growth factor (IGF-I) IA and IB gene, exon 1, Accession Nos. M12659, M77496; Rat insulin-like growth factor I (IGF-I) mRNA, Accession No. M15480; Chicken insulin-like growth factor (IGF-I) mRNA, Accession Nos. M3279 1, M29720; Salmon insulin-like growth factor I (IGF-I) mRNA, M32792; X. laevis insulin-like growth factor I (IGF-I) mRNA, Accession No. M29857. Thus, the invention should be construed to include DNA encoding IGF-I, or a biologically active portion thereof, from humans and non-human mammals, including but not limited to mouse, human, cow, pig, horse, sheep, rat, goat, Xenopus, and the like, which IGF-I functions in a substantially similar manner to the rat IGF-I described herein. Furthermore, IGF-I is thought to serve a similar role in other vertebrates as it does in mammals. Thus, transgenic fish or birds having and expressing IGF-I in mature muscle fibers (i.e., via the use of muscle specific promoters) will result in increased muscle mass in juvenile and adult animals. In preliminary experiments using bird muscle cells in vitro, it is evident that IGF-I has an effect on these cells, which effect is predictive that treatment of avian, fish and other muscle cells with the appropriate IGF-I will serve to increase the mass and strength of muscle tissue in these animals.

The term "non-human transgenic vertebrate animal" as used herein means an animal, the somatic and germ cells of which comprise an isolated nucleic acid comprising IGF-I.

Preferably, the nucleotide sequence comprising the gene encoding IGF-I is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the gene encoding human IGF-I and whose sequence is provided in (1986, J. Biol. Chem. 261:4828-4832). It should be noted that alternative RNA processing in the cell results in the production of two IGF-I precursor polypeptides and that both forms work in the experiments described herein. In addition, IGF-I obtained form rat liver or muscle may be used for comparison (1987, Mol. Endocrinol. 1:243-248).

The use of the term "DNA encoding" should be construed to include the DNA sequence which encodes the desired protein and any necessary 5' or 3' untranslated regions accompanying the actual coding sequence.

The term "expression of a nucleic acid" as used herein means the synthesis of the protein product encoded by the nucleic acid.

By "biologically active" as used herein, is meant an IGF-I protein, or any portion, modification or variant thereof, which is capable of mediating a substantially similar increase in and/or prevention of the loss of muscle mass and specific muscle strength as demonstrated for rat IGF-I as measured by the methods described herein.

By the term "specific strength," as used herein, is meant the force generated by a muscle divided by its cross-sectional area as determined pursuant to the methods described by Brooks and Faulkner (1988, J. Physiol. 404:71-82).

The invention should also be construed to include DNA encoding variants of IGF-I which retain IGF-I biological activity as defined previously herein. Such variants, i.e., analogs of proteins or polypeptides of IGF-I, include proteins or polypeptides which have been or may be modified using recombinant DNA technology such that the protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to, variants conferring enhanced stability on the protein in muscle, enhanced specific activity of the protein, and increased ability of the protein to remain localized in muscle tissues. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutaric acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine;

phenylalanine, tyrosine.

Preferably, the amino acid sequence of an IGF-I analog is about 70% homologous, more preferably about 80% homologous, even more preferably about 90% homologous, more preferably, about 95% homologous, and most preferably, at least about 99% homologous to the amino acid sequence of IGF-I described in (1986, J. Biol. Chem. 261:4828-4832) and in (1987, Mol. Endocrinol. 1:243-248).

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3' ATTGCC 5' and 3' TATGCG 5' share 50% homology.

Any number of procedures may be used for the generation of mutant or variant forms of IGF-I. For example, generation of mutant forms of IGF-I which do not circulate in the plasma of rAAV-IGF-I-injected animals may be accomplished by introducing deletion, substitution or insertion mutations into an IGF-I encoding nucleic acid residing on a plasmid template using ordinary recombinant DNA methodology described in any molecular biology manual, for example, methods described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, Green & Wiley, New York). Mutant IGF-I so generated is expressed and the resulting protein is assessed for its ability to remain localized in the muscle as demonstrated by a lack of significant increase in plasma levels of IGF-I such as that described herein. Mutant proteins which appear to be localized to the muscle tissues are then tested for IGF-I biological activity as defined herein. DNA encoding a mutated IGF-I protein which does not cause a significant rise in IGF-I plasma levels but which retains IGF-I biological activity, is suitable for use in the rAAV vector of the invention.

Procedures for the introduction of amino acid changes in a protein or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in Sambrook et al, supra, and Ausubel et al., supra.

It is desirable but not essential that the nucleic acid encoding IGF-I which is introduced into muscle cells in a mammal be expressed and that the protein remain localized within the muscle tissue. This is because if higher than physiologically normal amounts of IGF-I enter the circulatory system, IGF-I may promote undesired growth of other cells which may result in detrimental rather than beneficial effects to the mammal into which IGF-I has been introduced. Thus, the present invention also relates to the use of a truncated form of IGF-I which prevents egress of IGF-I from the muscle tissue into the circulatory system. Such a truncated form is described, for example, in Sara et al. (1986, Physiol. Rev. 70:591-614), wherein there is disclosed a truncated form of IGF-I which is missing the three terminal amino acids in the mature IGF-I peptide.

Accordingly, in a preferred embodiment, the nucleic acid encoding IGF-I introduced into the muscle cells encodes a truncated version of IGF-I which is expressed therefrom. The truncated version of the full-length rat IGF-I cDNA disclosed herein is preferably missing the three terminal amino acids in the mature IGF-I peptide. Expression in muscle of the truncated version does not cause measurable increase in plasma levels of IGF-I in mice as detected by standard methods as set forth herein.

In a preferred embodiment, the invention relates to the use of a muscle specific promoter/regulatory region, i.e., the MLC 1/3 promoter, such that DNA encoding IGF-I operably linked to the promoter/regulatory region is expressed at highest levels in type IIb/x fibers. However, the invention is not limited to the use of this or any other particular promoter/regulatory region to drive expression of the DNA encoding IGF-I. Indeed, other promoter/regulatory regions capable of driving expression of a heterologous gene to high levels in muscle cells, more desirably, in type IIb/x muscle cells, include, but are not limited to, the skeletal .alpha.-actin promoter and the muscle creatine kinase promoter/enhancer, and the like. Other muscle specific promoters may be used, including the skeletal muscle troponin subunit promoter, such as the fast troponin C promoter/enhancer sequence. It is important to note that it is preferable to use a muscle specific promoter when administering IGF-I to an animal, although it is possible to use a variety of non-muscle specific promoters as well, including constitutive promoters such as viral promoters. However, when the invention includes expression of IGF-I in a transgenic animal, a muscle specific promoter is the promoter of choice, in order to avoid any potential deleterious side effects which may arise as a result of expression of IGF-I in non-muscle tissue.

In another preferred embodiment, the rAAV-IGF-I recombinant of the invention comprises several DNA elements. These DNA elements include at least two copies of an AAV inverted terminal repeat (ITR) sequence, a 1.5 kb MLC 1/3 promoter/regulatory element previously discussed herein, a 0.85 kb SV40 polyadenylation sequence signal, and a 0.9 kb MLC 1/3 enhancer element, all operably linked to the IGF-I DNA coding region, as well as any necessary 5' or 3' untranslated regions which flank the DNA encoding IGF-I. However, the invention should not be interpreted as being limited to the presence of any or all of these particular elements or to any particular arrangement thereof. Rather, the invention encompasses other promoter/regulatory regions, enhancers, polyadenylation signal sequences, and the like, arranged in various orders and permutations thereof. Further, the invention includes constructs which do not have one or more of the above-stated DNA elements.

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

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

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

The rAAV vector of the invention also comprises 5' and 3' untranslated regions of DNA which flank the 0.6 kb rat IGF-I cDNA sequence. In the rAAV-IGF-I vector exemplified in the experimental examples section, the 5' untranslated region flanking the IGF-I sequences is as follows: At the 5' end of the IGF-I sequences, the 1.5 kb MLC 1/3 promoter sequence.

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

In the rAAV-IGF-I vector exemplified in the experimental details section, the 3' untranslated region flanking the IGF-I sequences is as follows: At the end of the translation stop signal, is the 0.85 kb SV40 poly A signal sequence which is followed by the 0.9 kb MLC 1/3 enhancer sequence.

It will be appreciated that other 5' and 3' untranslated regions of DNA may be used in place of those recited in the case of IGF-I.

The rAAV vector of the invention also comprises a transcription termination signal. While any transcription termination signal may be included in the vector of the invention, preferably, the transcription termination signal is the SV40 transcription termination signal.

In a preferred embodiment, a muscle enhancing dose of about 1010 plaque-forming units (pfu) of rAAV-IGF-I recombinant was administered to the muscle tissue of mice.

By the term "muscle enhancing dose" as the term is used herein, is meant a dose of an isolated nucleic acid, preferably but not necessarily in a recombinant virus vector, wherein the isolated nucleic acid encodes a mammalian IGF-I gene, wherein the isolated nucleic acid is administered to a muscle and IGF-I expressed therefrom induces an increase in muscle size and muscle strength compared with muscle to which an isolated nucleic acid is not administered or with a muscle to which a similar isolated nucleic acid dose is administered which either does not encode or does not express IGF-I.

By the term "administered" as used herein, is meant any method for the introduction of an isolated nucleic acid into muscle tissue. One preferred method is injection of the nucleic acid, or a recombinant vector comprising the nucleic acid either directly into the muscle or into the interstitial space surrounding the muscle of a vertebrate animal. Alternatively, systemic delivery of viral vectors or any other means by which isolated nucleic acid can be introduced into muscle cells including "gene gun", liposomes, and the like are also included in the invention.

The invention is not limited solely to the delivery of factors for the treatment of aging-associated loss of muscle mass and function. Rather, the invention should be construed to include a variety of vectors encoding other muscle enhancing factors for the treatment of various muscle disorders or conditions, which factors may be delivered using the methods of the present invention to the muscle cells of a mammal. Thus, the invention should be construed to include delivery of fibroblast growth factor, neurotrophins, and the like, in combination with IGF-I, or delivery of IGF-I alone to vertebrates.

The invention should also be construed to include nucleic acids encoding various proteins which are useful for the treatment of other muscle associated disease states in a mammal. Such muscle associated disease states include, but are not limited to, Becker muscular dystrophy, Duchenne muscular dystrophy, Limb Girdle muscular dystrophy, facioscapulohumeral dystrophy, spinal muscular dystrophy and amyotrophic lateral sclerosis.

By "therapeutic effect" as used herein as it relates to IGF-I, is meant any increase in muscle mass and muscle strength and/or prevention of loss of muscle mass and muscle strength caused by senescence, disease, weightlessness, or any other condition which causes decrease in muscle mass or strength.

According to the invention, it has been discovered that a preparation of an rAAV vector comprising nucleic acid encoding IGF-I injected into the muscle tissue of an animal at a single site per dose increases muscle mass and strength in young animals and prevents loss of muscle mass and strength in old animals. However, the invention is not limited to this injection regimen. Rather, treatment regimens which are contemplated include a single dose or dosage which is administered hourly, daily, weekly or monthly, or yearly.

The route of administration of the vaccine may also vary depending upon the disorder to be treated.

Typically, the number of viral vector genomes per gram of muscle which are administered in a single injection ranges from about 109 to about 1013. More preferably, the number of viral vector genomes/mammal which are administered in a single injection is about 2x1010 viral genomes per gram of muscle.

When the method of the invention comprises multiple site simultaneous injections, or several multiple site injections comprising injections into different muscle sites over a period of several hours (for example, from about less than one hour to about two or three hours), the total number of viral vector genomes administered is identical to that recited in the single site injection method.

For administration of the rAAV vector of the invention in a single site injection, a suspension of virus is injected directly into the muscle. For multiple site injection, a needle is inserted into the muscle tissue of the mammal. The vector is injected essentially continuously along the needle track so that a series of intramuscular sites are injected with each injection, each site therefore being at a position further into the muscle tissue than the previous site. Each injection targets from about 5 to about 30 sites along the needle track and patients may receive about 50 total injections. The procedure is therefore akin to an acupuncture procedure which is preferably carried out under anesthesia.

Multiple site injection of rAAV may also be accomplished using a multiple injection device such as that commonly used for the detection of tuberculosis infection.

In a preferred embodiment, the rAAV-IGF-I vector is suspended in a solution of 10% glycerol/HBS. However, the invention is not limited to this formulation. Rather, for administration to the mammal, the rAAV vector comprising IGF-I may be suspended in any pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8. Other pharmaceutically acceptable carriers which are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.

The rAAV vector of the invention may also be provided in the form of a kit, the kit comprising, for example, a freeze-dried preparation of vector in a dried salts formulation, sterile water for suspension of the vector/salts composition and instructions for suspension of the vector and administration of the same to the mammal.

Thus, included in the invention is a kit for enhancing or preventing the loss of muscle mass and strength. The kit comprises a muscle enhancing dose of an isolated nucleic acid encoding an IGF-I, or a biologically active portion thereof, and/or a recombinant vector comprising the nucleic acid, and an applicator for administering the nucleic acid to muscles, and instructions for using the kit. The instructions for using the kit depend on the particular human or veterinary patient for which the kit is to be used and the target muscle site(s).

In a preferred embodiment, the instructions comprise directions on how to administer a rAAV-IGF-I recombinant to the hind limb of mice. These instructions simply embody the examples provided herein.

The kit may also include instructions on how to administer the rAAV-IGF-I recombinant using the applicator provided therewith.

By the term "applicator" as the term is used herein, is meant any device including but not limited to a hypodermic syringe, a gene gun, and the like, for administering the DNA encoding IGF-I, or a biologically active portion thereof, into the muscles of a human or veterinary patient.

The compositions and methods described herein have the following applications. Muscle strength in aging humans may be preserved and/or enhanced; injured muscle may be caused to heal more effectively and more rapidly; muscle mass may be controlled during disease and/or during prolonged stays in reduced gravity environments; cosmetic body sculpting may be possible by promoting muscle hypertrophy in selected muscle tissue; muscle growth in young animals may be enhanced; muscle mass in adult animals may be enhanced; and, in diabetes, the compositions and methods may be useful for the promotion of glucose clearance from the muscle tissue.

IGF-I is also known to promote glucose uptake in skeletal muscle in an insulin-independent manner, and thus, also be of use in controlling blood glucose levels in diabetics.

In addition as noted herein, the invention includes a non-human transgenic vertebrate animal comprising an isolated nucleic acid comprising IGF-I, which IGF-I is preferably operably linked to a muscle specific promoter/regulatory region at its 5' end, and also comprises a polyadenylation signal at its 3' end. Such non-human transgenic vertebrate animals are expected to comprise up to or even more than 50% more muscle mass than their otherwise identical non-transgenic counterparts. In addition, the muscle mass of the transgenic animal progresses much faster than that of an otherwise identical non-transgenic counterpart animal. Thus, when used as a source of food, the transgenic animal of the invention may be brought to market faster than the non-transgenic counterpart animal. The generation of transgenic animals is well known in the art and is described, for example, in Hogan et al. (1986, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor, N.Y.).

Claim 1 of 13 Claims

What is claimed is:

1. An isolated nucleic acid comprising a vertebrate Insulin-like Growth Factor I (IGF-I) coding region, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5' side by an SV40 intron sequence and wherein said IGF-I coding region is flanked on the 3' end by an SV40 polyadenylation signal sequence.




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