Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
  • C12N2810/6027—Vectors comprising as targeting moiety peptide derived from defined protein from viruses ssDNA viruses

Definitions

• the present invention relates to compositions, methods and kits for systemic nucleic acid sequence delivery.
• the present invention relates to systemic nucleic acid sequence delivery without conventional systemic administration aids (SAAs).
• SAAs systemic administration aids
• vascular permeability agents such as VEGF
• AAV adeno-associated virus
• the present invention also provides methods of treating disease by co-administration of nucleic cid sequences encoding Igf-1 and dystrophin or dystrophin-like proteins.
• compositions and methods that allow for systemic nucleic acid sequence delivery without the need for systemic administration aids such as increased vascular pressure, isolated blood vessels or organs, or extracorporeal support for a subject.
• the present invention provides compositions, methods and kits for systemic nucleic acid sequence delivery.
• the present invention provides systemic nucleic acid sequence delivery without conventional systemic administration aids (SAAs).
• SAAs systemic administration aids
• vascular permeability agents such as VEGF
• AAV recombinant adeno-associated virus
• the present invention also provides methods of treating disease by co-administration of nucleic cid sequences encoding Igf-1 and dystrophin or dystrophin-like proteins.
• the present invention provides methods of systemic transduction of extravascular tissue comprising; a) providing; i) a first composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise a nucleic acid sequence of interest, ii) a second composition comprising a vascular permeabilizing agent (e.g. a VEGF molecule such as a VEGF isoform), and iii) a subject comprising a first type of extravascular tissue; and b) administering the first and second compositions systemically to the subject, without using a systemic administration aid (i.e. systemic administration aid- free administration), under conditions such that the first type of extravascular tissue is transduced by the nucleic acid vectors.
• a systemic administration aid i.e. systemic administration aid- free administration
• the first and second compositions are mixed together prior to the administering.
• the present invention provides methods of systemic transduction of extravascular tissue comprising; a) providing; i) a first composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise a nucleic acid sequence of interest, ii) a second composition comprising a vascular permeabilizing agent, and iii) a subject comprising a first type of extravascular tissue; and b) administering the first and second compositions systemically to the subject under conditions such that: i) the subject lacks at least one subject systemic administration aid; and ii) the first type of extravascular tissue is transduced by the nucleic acid vectors.
• the first and second compositions are mixed together prior to the administering.
• the subject lacks at least one subject systemic administration aid selected from the group consisting of: a reduction in body temperature of the subject; the subject is unconscious, the subject is on extracorporeal circulatory support, the first type of extravascular tissue is isolated from the remainder of the subject, the first type of extravascular tissue in the subject is exsanguinated, and the subject has hepatic inflow occlusion.
• the subject lacks at least two, or three or four or five subject systemic administration aids selected from the group consisting of: a reduction in body temperature of the subject; the subject is unconscious, the subject is on extracorporeal circulatory support, the first type of extravascular tissue is isolated from the remainder of the subject, the first type of extravascular tissue in the subject is exsanguinated, and the subject has hepatic inflow occlusion.
• the subject lacks all six of the following subject systemic administration aids: a reduction in body temperature of the subject; the subject is unconscious, the subject is on extracorporeal circulatory support, the first type of extravascular tissue is isolated from the remainder of the subject, the first type of extravascular tissue in the subject is exsanguinated, and the subject has hepatic inflow occlusion.
• the subject is conscious.
• the body temperature of the subject is normal (not reduced).
• the subject is free from extracorporeal circulatory support.
• the subject is not in cardiac arrest and/or is not under anesthesia.
• the first type of extravascular tissue is not isolated from the remainder of the subject.
• the first type of extravascular tissue is non-exsanguinated.
• the subject does not have heppatic inflow occlusion.
• the administering is performed without at least one blood vessel systemic administration aid.
• the present invention provides methods of systemic transduction of extravascular tissue comprising; a) providing; i) a first composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise a nucleic acid sequence of interest, ii) a second composition comprising a vascular permeabilizing agent, and iii) a subject comprising a first type of extravascular tissue; and b) administering the first and second compositions systemically to one or more blood vessels in the subject, without using at least one blood vessel systemic administration aid, under conditions such that the first type of extravascular tissue is transduced by the nucleic acid vectors.
• the first and second compositions are mixed together prior to the administering.
• the at least one blood vessel systemic administration aid is selected from the group consisting of: increased perfusion pressure in the one or more blood vessels (i.e. increased perfusion pressure not caused by the injection itself and not caused as a result of a biological reaction caused by the VPA); where one or more blood vessels are isolated from the remainder of the subject; wherein the site or sites of administration is not distant from the first type of extravascular tissue; wherein the administration is directly in the coronary circulation of the subject; wherein the administration is followed by administering an oxygen-transporting agent to the on or more blood vessels; and prior to the administration, delivering a vasodilating agent to the one or more blood vessels.
• the administering is conducted without at least two, or three, or four, or five blood vessel systemic administration aids selected from the group consisting of: increased perfusion pressure in the one or more blood vessels (i.e. increased perfusion pressure not caused by the injection itself and not caused as a result of a biological reaction caused by the VPA); wherein the one or more blood vessels are isolated from the remainder of the subject; wherein the site or sites of administration is not distant from the first type of extravascular tissue; wherein the administration is directly in the coronary circulation of the subject; wherein the administration is followed by administering an oxygen-transporting agent to the on or more blood vessels; and prior to the administration, delivering a vasodilating agent to the one or more blood vessels.
• increased perfusion pressure in the one or more blood vessels i.e. increased perfusion pressure not caused by the injection itself and not caused as a result of a biological reaction caused by the VPA
• the one or more blood vessels are isolated from the remainder of the subject
• the site or sites of administration is not distant from the first type of
• the administering is conducted without any of the following six blood vessel systemic administration aids: increased perfusion pressure in the one or more blood vessels (i.e. increased perfusion pressure not caused by the injection itself and not caused as a result of a biological reaction caused by the VPA), wherein the one or more blood vessels are isolated from the remainder of the subject, wherein the site or sites of administration is not distant from the first type of extravascular tissue, wherein the administration is directly in the coronary circulation of the subject, wherein the administration is followed by administering an oxygen-transporting agent to the on or more blood vessels, and prior to the administration, delivering a vasodilating agent to the one or more blood vessels.
• increased perfusion pressure in the one or more blood vessels i.e. increased perfusion pressure not caused by the injection itself and not caused as a result of a biological reaction caused by the VPA
• the one or more blood vessels are isolated from the remainder of the subject, wherein the site or sites of administration is not distant from the first type of extravascular tissue
• the administration is directly in the coronary
• the administering is in a blood vessel or blood vessels of the subject that is (are) distant from the first type of extravascular tissue (e.g. in a blood vessel not directly associated with the first type of extravascular tissue, or at least 5 inches, or 10 inches or 20 inches or 2 feet from the first type of extravascular tissue).
• the administering is to one or more blood vessels under physiologically normal (non-enhanced) pressure, and/or the pressure is not increased after the administering.
• the one or more blood vessels are not isolated from the remainder of the subject.
• the administration is not directly in the coronary circulation of the subject.
• the administering is not followed by administering an oxygen-transporting agent to the one or more blood vessels (e.g. no oxygen-transport agent is administered to the one or more blood vessels within 24- 48 hours of the administration of the first and second compositions, i other embodiments, no vasodilating agent is delivered to the one or more blood vessels prior, or during, to the administering the first and second compositions (e.g. no vasodilating agent is present in the first or second composition and no vasodilating agent is delivered before the first and second compositions are administered).
• at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or substantially all cells in the first type of extravascular tissue express the nucleic acid sequence of interest.
• the level of transduction is achieved with a single administration of the first and second compositions (e.g. no additional treatments are given over the next 90 days).
• the first and second compositions are administered simultaneously (e.g. as part of single combined-composition or within 20 seconds of each other).
• the first composition is administered within 5 minutes of the second composition, h some embodiments, the first composition is administered within 3 minutes, 2 minutes, 30 second, 10 seconds, 5 seconds, or at the same time as the second composition.
• the first composition and the second composition are administered to the same blood vessel.
• the first composition and the second composition are administered to different blood vessels, i additional embodiments, the first and/or second composition further comprises heparin.
• the methods further comprise providing a third composition comprising heparin, wherein the administering further comprises systemically administering the third composition to the subject.
• the first type of extravascular tissue is heart tissue (e.g. the heart or a portion thereof, such as the muscle tissue in the heart).
• the first type of extravascular tissue is skeletal muscle tissue (e.g. muscles in the legs, muscles in the arms, muscles in the abdomen, muscles in the back, muscles in the face, or substantially all the skeletal muscles of the subject).
• a significant proportion of the skeletal muscle tissue in the subject is transduced by the nucleic acid vectors (e.g.
• the nucleic acid sequence of interest expresses the nucleic acid sequence of interest
• substantially all of the skeletal muscle tissue in the subject is transduced by the nucleic acid vectors (e.g. as evidenced by at least 75 or at least 85%, or 90% of the skeletal muscle tissue expressing the nucleic acid sequence of interest). This may be determined by biopsy or inferred based on activity levels of the muscles before and after administering the first and second compositions.
• the nucleic acid sequence of interest encodes dystrophin or is a mini-dystrophin nucleic acid sequence.
• the nucleic acid vectors are viral vectors.
• the nucleic acid vectors are adeno-associated vectors.
• the adeno-associated vectors are AAV1-AAV9, (e.g. AAV6 or AAVl are preferred).
• the nucleic acid vectors comprise an AAV6 capsid (e.g., AAV6 vectors or AAV6 psuedo-typed vectors).
• the vascular permeabilizing agent is a VEGF molecule (e.g.
• a VEGF isoform or mimetic, VEGF165, PGF, fragmets thereof, an agent that is able to bind Fltl or Flkl, or a non-toxic vascular penneabilizing agent).
• the first and second compositions are together in a combined-composition.
• a vascular permeabilizing agent is conjugated to the nucleic acid vectors, hi other embodiments, the viral vectors further comprise a ligand specific for the vascular pemerabilizing agent (e.g. Fab, single chain antibody region, protein A, Fltl, Flkl, etc.).
• the first composition is a vasodilating agent-free composition (i.e.
• the second composition is a vasodilating agent-free composition (i.e. does not contain a vasodilating agent).
• the administering is conducted without ever administering a vasodilating agent.
• the administering is conducted without ever administering histamine.
• the subject has symptoms of a disease, and wherein the administering reduces at least one of the symptoms of disease. In some embodiments, this reduction in symptoms is the result of a single administration of the first and second compositions (e.g. no additional administration occurs over the 90 days following the initial administration).
• the disease is selected from heart disease, a muscular dystrophy, a nueuropathy such as Alzheimer's disease, hemophelia, cancer (e.g. breast cancer, lung cancer, skin cancer, prostate cancer, etc.), Pompe's disease, Fabry's disease, bacterial or viral infection, or diseases associated with aging.
• the subj ect has symptoms of disease in the first type of extravascular tissue, and wherein the administering reduces at least one of the symptoms of disease in the first type of extravascular tissue, hi other embodiments, the subject has symptoms of disease in the first type of extravascular tissue such that the first type of extravascular tissue has a function deficit compared to wild type (e.g.
• the administering at least partially compensates for the function deficit (e.g. reduces symptoms of disease is the exfravascular tissue, and/or improves the extravascular tissue such that it is nearly equal in function compared to the wild type; e.g. 80% of the activity of wild type or 90%, or 95% of the activity of wild type).
• the disease treated is a muscular dystrophy such as DMD.
• the first type of extravascular tissue is an organ selected from: heart, kidney, eyes, pancrease, liver, gall bladder, intestines, stomach, lungs, urinary track, and diaphram.
• the first type of extravascular tissue is skelatal muscle tissue, heart muscle tissue, diaphram tissue, leg muscle tissue, arm muscle tissue, etc.
• the subject comprises a second extravascular tissue type, and the administering is under conditions such that the first and second extravascular tissues are transduced by the nucleic acid vectors, hi preferred embodiments, the administering is performed with a needle and syringe. In particular embodiments, the administering is in one or more blood vessels of the subject.
• the nucleic acid sequence of interest is selected from dystrophin, mini-dystrophin, or other therapeutic sequences, hi further embodiments, the administering is conducted with a muscle transduction aid (e.g., heat, moderate exercise, ultrasound, etc.).
• the first composition comprises less than 1 x 10 nucleic acid vectors per milliliter (e.g., 8 x 10 11 nucleic acid vectors per milliliter or less; 1-5 x 10 11 nucleic acid vectors per milliliter or less; l x l 0 9 - 8 x 10 11 nucleic acid vectors per milliliter, or 1 x 10 10 - 3 x 10 11 nucleic acid vectors per milliliter).
• 19 the first composition is administered to the subject at a dosage of less than 1 x 10 vectors per kilogram of the subject (e.g., 8 x 10 11 vectors per kilogram of the subject or less; 1-5 x 10 11 vectors per kilogram of the subject or less; l x l 0 9 - 8 x 10 11 vectors per kilogram of the subject, or 1 x 10 10 - 3 x 10 11 vectors per kilogram of the subject).
• a dosage of less than 1 x 10 vectors per kilogram of the subject e.g., 8 x 10 11 vectors per kilogram of the subject or less; 1-5 x 10 11 vectors per kilogram of the subject or less; l x l 0 9 - 8 x 10 11 vectors per kilogram of the subject, or 1 x 10 10 - 3 x 10 11 vectors per kilogram of the subject.
• the compositions of the present invention comprises about 1 x 10 12 nucleic acid vectors per milliliter, about 1 x 10 13 nucleic acid vectors per milliliter, about 1 x 10 14 nucleic acid vectors per milliliter, about 1 x 10 15 nucleic acid vectors per milliliter, about 1 x 10 16 nucleic acid vectors per milliliter, or about 1 x 10 17 nucleic acid vectors per milliliter.
• the compositions contain no more than 1 x 10 16 nucleic acid vectors per milliliter, or 1 x 10 17 nucleic acid vectors per milliliter.
• compositions of the present invention are administered to a subject at a dosage of about 1 x 10 11 nucleic acid vectors per kilogram of subject, about 1 x 10 nucleic acid vectors per kilogram of subject, about 1 x 10 nucleic acid kilogram of subject, about 1 x 10 14 nucleic acid vectors per kilogram of subject, about 1 x 10 15 nucleic acid vectors per kilogram of subject, about 1 x 10 16 nucleic acid vectors per kilogram of subject, or about 1 x 10 17 nucleic acid vectors per kilogram of subject (e.g. about 4 x 10 14 nucleic acid vectors per kilogram of subject, or about 8 x 10 14 nucleic acid vectors per kilogram of subject).
• compositions contain no more than 1 x 10 nucleic acid vectors per kilogram of subject, or no more than 1 x 10 nucleic acid vectors per kilogram of subject.
• the administering is in a blood vessel in a limb (e.g. arm or leg).
• the administering is in a blood vessel of the subject that is distant from the first type of extravascular tissue (e.g. in a blood vessel not directly associated with the first type of extravascular tissue, or at least 5 inches, or 10 inches or 20 inches or 2 feet from the first type of extravascular tissue).
• the nucleic acid vectors are less than 70 ⁇ m in diamter (e.g., vectors with a diameter around lOnm to 50nm or 5nm to 70nm).
• the present invention provides methods of systemic transduction of extravascular tissue comprising; a) providing; i) a composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise an AAV6 capsid and a nucleic acid sequence of interest, and ii) a subject comprising a first type of extravascular tissue; and b) administering the composition systemically to the subject, without using a systemic administration aid, under conditions such that the first type of extravascular tissue is transduced by the nucleic acid vectors.
• the present invention provides methods of systemic transduction of extravascular tissue comprising; a) providing; i) a composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise an AAV6 capsid and a nucleic acid sequence of interest, and ii) a subject comprising a first type of extravascular tissue; and b) administering the composition systemically to the subject under conditions such that: i) the subject lacks at least one subject systemic transduction aid; and ii) the first type of extravascular tissue is transduced by the nucleic acid vectors.
• the present invention provides methods of systemic transduction of extravascular tissue comprising; a) providing; i) a composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise an AAV6 capsid and a nucleic acid sequence of interest, and ii) a subject comprising a first type of extravascular tissue; and b) administering the composition systemically to one or more blood vessels in the subject, without using at least one blood vessel systemic administration aid, under conditions such that the first type of extravascular tissue is transduced by the nucleic acid vectors.
• the composition comprises at least 1 x 10 nucleic acid 1 • 19 vectors per milliliter (e.g., 8 x 10 nucleic acid vectors per milliliter or more, 1-5 x 10 nucleic acid vectors per milliliter or more; 1 x 10 - 8 x 10 nucleic acid vectors per milliliter; 1 x 10 13 - 3 x 10 13 nucleic acid vectors per milliliter).
• the composition is administered to the subject at a dosage of greater than 1 10 vectors per kilogram of the subject (e.g., 8 x 10 13 vectors per kilogram of the subject or more, 1-5 x 10 13 vectors per kilogram of the subject or more; 1 x 10 13 - 8 x 10 13 vectors per kilogram of the subject, or 1 x 10 - 3 x 10 vectors per kilogram of the subject).
• 1 10 vectors per kilogram of the subject e.g., 8 x 10 13 vectors per kilogram of the subject or more, 1-5 x 10 13 vectors per kilogram of the subject or more; 1 x 10 13 - 8 x 10 13 vectors per kilogram of the subject, or 1 x 10 - 3 x 10 vectors per kilogram of the subject.
• the present invention provides methods of systemic transduction of extravascular tissue comprising; a) providing; i) a first composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise a nucleic acid sequence of interest, and wherein the first composition comprises less than 1 x 10 12 nucleic acid vectors per milliliter, ii) a second composition comprising a vascular permeabilizing agent, and iii) a subject comprising a first type of extravascular tissue; and b) administering the first and second compositions systemically to the subject under conditions such that the first type of extravascular tissue is transduced by the nucleic acid vectors.
• the present invention provides methods of systemic transduction of extravascular tissue comprising; a) providing; i) a composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise an AAV6 capsid and a nucleic acid sequence of interest, and wherein the composition comprises at least 1 x 10 nucleic acid vectors per milliliter, and ii) a subject comprising a first type of extravascular tissue; and b) administering the composition systemically to the subject under conditions such that the first type of extravascular tissue is transduced by the nucleic acid vectors.
• the present invention provides compositions comprising: a) a vascular permeabilizing agent, and b) nucleic acid vectors comprising a nucleic acid sequence of interest, wherein the nucleic acid vectors are present in a concentration between 1 x 10 9 nucleic acid vectors and 1 x 10 12 nucleic acid vectors per milliliter.
• the nucleic acid vectors are present in a concentration between 1 x 10 10 nucleic acid vectors and 5 x 10 11 nucleic acid vectors per milliliter.
• the nucleic acid vectors are present in a concentration of about 1 x 10 11 nucleic acid vectors per milliliter.
• the compositions do not contain a vasodilating agent. In other embodiments, the composition does not contain histamine. In some embodiments, the compositions further comprise heparin. In prefened embodiments, the vascular permeabilizing agent is non-toxic. In particular embodiments, the vascular permeabilizing agent is conjugated to the nucleic acid vector. In preferced embodiments, the nucleic acid sequence of interest is dystrophin, mini-dystrophin sequence, or a truncated or modified dystrophin sequence. In some embodiments, the nucleic acid vectors are viral vectors.
• the nucleic acid vectors are adeno-associated vectors (e.g., AAV1-AAV9, AAV6 or AAVl).
• the nucleic acid vectors comprise an AAV6 capsid (e.g., AAV6 vectors or AAV6 psuedo-typed vectors).
• the vascular permeabilizing agent is a VEGF molecule.
• kits comprising; a) a first composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise a nucleic acid sequence of interest, b) a second composition comprising a vascular permeabilizing agent, and c) a written insert comprising instructions for systemically administering the first and second compositions, wherein the instructions do not indicate that one or more systemic administration aids are to be employed.
• kits comprising: a) a composition comprising: i) nucleic acid vectors, wherein the nucleic acid vectors comprise a nucleic acid sequence of interest, and ii) a vascular permeabilizing agent, and c) a written insert comprising instructions for systemically administering the composition, wherein the instructions do not indicate that one or more systemic administration aids are to be employed.
• kits comprising; a) a first composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise an AAV6 capsid and a nucleic acid sequence of interest, and b) a written insert comprising instructions for systemically administering the first and second compositions, wherein the instructions do not indicate that one or more systemic administration aids are to be employed.
• the present invention provides methods of treating a subject comprising; a) providing; i) a first plurality of nucleic acid sequences encoding dystrophin or dystrophin-like proteins, ii) a second plurality of nucleic acid sequences encoding insulin-like growth factor 1 (Igf-1), and iii) a subject comprising muscle tissue; and b) administering the first and second plurality of nucleic acid sequences to the subject under conditions such that the muscle tissue of the subject exhibits an increase in mass, strength, and protection from contraction-induced injury.
• Igf-1 insulin-like growth factor 1
• the dystrophin-like proteins are selected from the group consisting of: micro-dystrophin proteins, utrophin proteins, micro-utrophin proteins, dystrophin/utrophin chimera proteins, and micro- dystrophin/utrophin chimera proteins.
• the administering comprises systemic injection of the first and second plurality of nucleic acid sequences simultaneously, or within 1 hour of each other, or within 12 days of each other (e.g. within 5 minutes to 7 days of each other).
• the first or second plurality of nucleic acid sequences are packaged in adeno-associated virus capsids.
• the subject is an elderly human (e.g.
• the present invention provides methods of treating a subject comprising; a) providing; i) a composition comprising nucleic acid vectors, wherein the nucleic acid vectors comprise a nucleic acid sequence encoding insulin-like growth factor 1 (Igf-1), and ii) a subject with symptoms of a muscle wasting disease; b) administering the composition to the subject under conditions such that at least one symptom of muscle wasting is reduced or eliminated, h prefereed embodiments, the subject is an elderly human (e.g. a human patient 65 years or older who's exhibits symptoms of muscle tissue wasting).
• Igf-1 insulin-like growth factor 1
• the present invention provides compositions comprising micro-utrophin proteins or micro-utrophin dyst hin chimeric proteins, or nucleic acid sequences encoding these proteins.
• the present invention provides nucleic acid sequences encoding micro-utrophin proteins with 4.0 utrophin spectrin-like repeats (see, e.g. Figure 12).
• the present invention provides nucleic acid sequences encoding micro-utrophin/dystrophin chimeric proteins with 4.0 spectrin-like repeats from utrophin and dystrophin (e.g.
• the micro proteins comprise other elements (e.g. from utrophin or dystrophin) such as an actin binding amino-terminal domain, 2, 3 or 4 hinge domains, and WW and cysteine rich domains, certain embodiments, the micro-utrohin sequences are similar to or identical to SEQ ID NOs. 1, 2 and 6.
• the micro-utrophin nucleic sequence or peptides are used to treat a subject (e.g. a subject with a muscle wasting disease).
• a vascular permeablizing agent e.g. VEGF- A or AAV6 empty capsids
• VEGF- A or AAV6 empty capsids are combined with the micro-utrophins nucleic acid sequences.
• FIGURES show a plate demonstrating the gray/black muscles of mice receiving vector and VEGF/heparin, as well as mice receiving vector alone.
• the dark shading is a reaction generated from the enzyme produced by the gene (LacZ) delivered by the virus.
• Figure 2 shows a graph demonstrating the enzymatic activity measured in muscles and various organs from mice receiving various doses of vector with and without co- treatment of VEGF and heparin.
• figure 2 shows /3-galactosidase activity (driven by the CMV promoter) in various muscles and organs following systemic delivery of 2 x 10 11 or 10 12 doses of viral vector with and without VEGF
• Figure 3 shows various muscles of the body transduced with a gene encoding ⁇ - galactosidase following a single i.v. injection (using the AAV6-CMV-LacZ constructs into a conscious animal). The gray staining is indicative of positive signal.
• Figure 4 shows a transverse section of a mouse heart harvested l id after a single systemic delivery of lxlO 12 viral genomes (i.e.
• FIG. 5 demonstrates a functional correction of dystrophic muscle (tibialis anterior) following a single systemic administration of lxl 0 12 viral genomes of pseudo- typed AAV6 CK6-micro-dystrophin.
• FIG 6 shows that dystrophic animals have extremely widespread expression of approximately wild-type levels of micro-dystrophin in the quadriceps (Quad) muscle following systemic delivery of pseudo-typed AAV6 CK6-micro-dystrophin.
• Figure 7 shows that dystrophic animals have extremely widespread expression of approximately wild-type levels of micro-dystrophin in the tibialis anterior following systemic delivery of pseudo-typed AAV6 CK6-micro-dystrophin.
• Figure 8 shows results from Example 2 where rAAV6/RSV-hpAP was injected into the jugular vein of a 2 month old beagle and heart and diaphragm tissue was examined for hpAP expression.
• Figure 9 is a graph that shows the results from Example 3 where rAAV6/CMV-lacZ plus VEGF-A121 were injected into the tail vein of mice and viral transduction in cardiac and skeletal muscle tissue was analyzed 11 days later for gal activity.
• Figure 10 shows the high level of beta-gal expression in cardiac tissue from the intravenous and intra-arterial administration of rAAV6-CMVlacZ described in Example 4 using different injection intervals (3 seconds, 60 seconds, or 600 seconds).
• Figure 11 shows results from Example 5, where preparation of approximately 95% empty AAV6 capsids ("column purified") were shown to enhance AAV6 viral transduction to a greater extent than a preparation of approximately 50% empty AAV6 viral capsids ('CsCl-banded").
• Figure 12 shows schematics of various micro-dystrophin, micro-utrophin, and micro-dystrophin/utrophin chimeras .
• Figure 13 A shows the nucleic acid sequence (SEQ ID NO:l) of the murine micro- utrophin sequence employed in Example 6.
• Figure 13B shows the nucleic acid sequence (SEQ ID NO:2) of an additional murine micor-utrophin sequence that could be employed to treat muscle wasting type conditions.
• Figure 13C shows the nucleic acid sequence (SEQ ID NO:6) of a human micro-utrophin sequence that could be employed (e.g. for treating a human subject). Of course changes could be introduced into these sequences to produce additional micro-utrophins sequences (see e.g. Figure 12).
• Figure 14A shows the micro-dystrophin and Igf-1 constructs used in Example 7.
• Figure 14B shows a graph reporting dystrophin positive area type results of Example 7.
• Figure 15 shows results for the mice treated in Example 7, including: A) muscle mass/body mass, B) tetanic force measurements; C) percentage force retention (LCI); and D) percentage force retention (LC2).
• Figure 16 shows a graph of results of tissue measurements from mice treated as described in Example 7, including: A) percentage of central nuclei; B) number of fibers/area; and C) fiber diameter.
• Figure 17A shows a graph that indicates that intravenous administration of rAAV6- Igf-1 vectors to old mice in Example 8 results in increased body mass consistent with increased muscle mass.
• Figure 17B show the change in Tibialis anterior (hindlimb) muscle mass for the control mice and mice receiving Igf-1 as described in Example 8, revealing that Igf-1 treatment in old mice results in increased muscle strength and performance.
• Figure 18A is a graph that shows the tibialis anterior (TA) muscles of old mice treated with Igf-1 expressing vectors in Example 8 generates increased force compared with the muscles of untreated mice.
• Figure 18B is a graph that shows that the muscles of Igf-1 treated mice in Example 8 display increased force output over a protocol of repetitive stimulation and at recovery.
• Figure 19 show the nucleic acid sequences for murine Igf-1 Eb (SEQ ID NO:3), murine Igf-I Ea (SEQ ID NO:4), and human Igf-1 (SEQ ID NO:5).
• the terms “subject” and “patient” refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human.
• the term “subject systemic administration aid” or “SSAA” refers to non-trivial changes that are made to a subject prior to or during systemic administration of a nucleic acid vector in order to increase the transduction efficiency of the administered nucleic acid vector.
• SSAAs include: a reduction in body temperature of the subject; a subject that is unconscious (e.g., subject to anesthesia), placing a subject on extracorporeal circulatory support (e.g., using a heart and lung machine), isolating the target extravascular tissue from the remainder of the subject, exsanguinating (removing the majority of blood from) the target extravascular tissue/organ, providing the subject with hepatic inflow occlusion.
• blood vessel systemic administration aid refers to: non-trivial changes that are made to a blood vessel prior to or during systemic administration of a nucleic acid vector; choice of blood vessel site of administration of the nucleic acid vector; or reagents applied to a blood vessel prior to, during, or immediately after administration; all used in order to increase transduction efficiency of the administered nucleic acid vector.
• BVSAAs include: increased perfusion pressure in a blood vessel (i.e.
• a site of administration that is close to and/or generally associated with the target extravascular tissue (i.e. not a site distant from the target extravascular tissue), administration directly in the coronary circulation of the subject (i.e. choosing a coronary blood vessel for site of administration); administering an oxygen-fransporting agent to one or more blood vessels following administration of the nucleic acid vector; and prior to the administration of the nucleic acid vectors, delivering a vasodilating agent to a blood vessel.
• systemic administration aid or "SAA” includes both subject systemic administration aids (SSAAs) and the blood vessel systemic administration aids (BVSAAs).
• fragment refers to a polypeptide that has an amino- terminal deletion, and/or carboxy-terminal deletion, and/or both amino terminal and carboxy terminal deletion (e.g. leaving only a center portion) as compared to the native protein, but where the remaining amino acid sequence is identical to the conesponding positions in the amino acid sequence deduced from a full-length cDNA sequence.
• Fragments typically are at least 4 amino acids long, preferably at least 20 amino acids long, usually at least 50 amino acids long or longer, and span the portion of the polypeptide required for intermolecular binding of the compositions with its various ligands and/or substrates.
• portion when in reference to a nucleotide sequence (as in "a portion of a given nucleotide sequence") refers to fragments of that sequence. The fragments may range in size from four nucleotides to the entire nucleotide sequence minus one nucleotide (10 nucleotides, 20, 30, 40, 50, 100, 200, etc.).
• vector or nucleic acid vector
• nucleic acid molecules e.g., plasmids
• viruses e.g. adeno-assoicated viruses
• vector examples include, but are not limited to plasmids, adeno- associated viruses, and adenoassociated viruses.
• nucleic acid vector When a nucleic acid vector contains a nucleic acid sequence of interest, generally the nucleic acid vector includes, for prokaryotic expression, nucleic acid sequences such as a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. If the nucleic acid vector is to be used in eukaryotic cells, nucleic acid sequence such as promoters, enhancers, and termination and polyadenylation signals are generally included with the nucleic acid sequence of interest. As used herein, the term “transduction” refers to the introduction of foreign nucleic acid, such as a nucleic acid sequence of interest, into cell via a nucleic acid vector.
• extravascular tissue refers to a tissue which is located outside of a blood vessel. It is noted that extravascular tissue may surround a given blood vessel. Examples of extravascular tissue include, but are not limited to, skeletal muscle, heart muscle, diaphram tissue, etc.
• vascular permeability agent is a composition of matter which, when supplied to a blood vessel of an animal, preferably a mammal, increases the permeability of the endothelial layer of the vessel, such that substances within the vessel may pass through the endothelial layer.
• a vasodilating agent is a composition of matter which, when supplied to a blood vessel of an animal, preferably a mammal, increases the luminal diameter of the vessel. Stated another way, a vasodilating agent, when administered to a blood vessel of an animal, increases the caliber of the vessel.
• the "perfusion pressure" within a blood vessel means the peak pressure differential between the fluid within the lumen of the vessel and the fluid sunounding the vessel. It is understood that the peak pressure within the vessel conesponds to the driving force for blood flow through the vessel by the beating action of the animal heart.
• the "normal physiological" perfusion pressure within a blood vessel means the perfusion pressure within the vessel of a healthy animal in a resting state.
• an "oxygen-transporting agent” means a composition of matter which, when in a liquid or solution form, is capable of capturing an oxygen molecule and delivering the oxygen molecule to a biological oxygen carrier such as hemoglobin or myoglobin.
• a biological oxygen carrier such as hemoglobin or myoglobin.
• numerous synthetic blood substitutes and perfluorochemical liquids are oxygen-fransporting agents.
• extracorporeal circulatory support means a subject is attached to a mechanical device which is capable of circulating the blood of the subject through all or a part of the circulatory system of the subject without assistance from the heart of the subject.
• a heart-lung machine which is well known in the art, is a device which is useful for providing extracorporeal circulatory support.
• the present invention provides compositions, methods and kits for systemic nucleic acid sequence delivery.
• the present invention provides systemic nucleic acid sequence delivery without conventional systemic administration aids (SAAs).
• a vascular permeability agents such as VEGF
• AAV adeno-associated virus
• the present invention also provides methods of treating disease by co-administration of nucleic cid sequences encoding Igf-1 and dystrophin or dystrophin-like proteins.
• the present invention provides methods and compositions for systemic nucleic acid sequence delivery to extravascular tissue such as skeletal muscle or heart muscle without using systemic administration aids (SAAs) such as subject systemic administration aids (SSAAs) and blood vessel systemic administration aids (BVSAAs).
• SAAs systemic administration aids
• SSAAs subject systemic administration aids
• BVSAAs blood vessel systemic administration aids
• Stedman teaches the necessity to isolate a blood vessel, treat with a vasodilating agent, and treat with a vascular pemeability agent prior to systemically administering any type of vector (as well as providing an oxygen-transporting agent to the vessel after administration).
• Stedman also shows, in Table 1, that muscle can only be transduced using, inter alia, moderate or high pressure in the blood vessel (as well as requiring both histamine and papaverine).
• the Stedman patent also indicates the need to place a subject on extracorporeal circulatory support and oxygenation prior to providing any vector (as well as requiring an increase in perfussion pressure in the blood vessel). The present invention does not require these elaborate and often dangerous procedures.
• the present invention (e.g. as shown in the Example below) also does not employ the high injection volumes and increases in vascular pressure that are used in many reported protocols and are considered to be a critical to their efficiencies. Indeed, in prefened embodiments, the present invention does not necessitate increases in blood pressure nor the replacement of the blood with a reagent-containing buffer. For example, in the Example below, injections directly into the blood represented only about 10% total blood volume; a clinically tolerated volume in humans. As noted above, the present invention allows for systemic nucleic acid administration, (with extravascular transduction), without the need for systemic administration aids, such as subject systemic administration aids or blood vessel administration aids.
• Subject systemic administration aids or "SSAAs" are non-trivial changes that are made to a subject prior to or during systemic administration of a nucleic acid vector in order to increase the transduction efficiency of the administered nucleic acid vector.
• the present invention allows systemic administration and transduction without the need for such aids.
• SSAAs include: a reduction in body temperature of the subject; a subject that is unconscious (e.g., subject to anesthesia), placing a subject on extracorporeal circulatory support (e.g., using a heart and lung machine), isolating the target extravascular tissue from the remainder of the subject, exsanguinating (removing the majority of blood from) the target extravascular tissue/organ, and providing the subject with hepatic inflow occlusion.
• the present invention allows systemic nucleic acid administration where: the body temperature of the subject is normal (not reduced); the subject is free from extracorporeal circulatory support and/or oxygenation; the subject is not in cardiac arrest and/or is not under anesthesia; the extravascular tissue is not isolated from the remainder of the subject; the extravascular tissue is non-exsanguinated; the subject does not have heppatic flow occlusion.
• Blood vessel systemic administration aids or BVSAAs are non-trivial changes that are made to a blood vessel prior to or during systemic administration of a nucleic acid vector; choice of blood vessel site of administration of the nucleic acid vector; or reagents applied to a blood vessel prior to, during, or immediately after administration; all used in order to increase transduction efficiency of the administered nucleic acid vector.
• the present invention allows systemic administration of nucleic acid vectors without BVSAAs.
• the BVSAAs include: increased perfusion pressure in a blood vessel; isolating the blood vessel from the remainder of the subject; employing a site of administration that is close to and/or generally associated with the target extravascular tissue (i.e.
• the present invention allows systemic nucleic acid administration where: the administering is in a blood vessel or blood vessels of the subject that is (are) distant from the target extravascular tissue (e.g.
• the administering is to one or more blood vessels under physiologically normal (non-enhanced) pressure, and/or the pressure is not increased after the administering; the one or more blood vessels are not isolated from the remainder of the subject; the administration is not directly in the coronary circulation of the subject; the administering is not followed by administering an oxygen- transporting agent to the on or more blood vessels (e.g.
• no oxygen-transport agent is administered to the one or more blood vessels within 24-48 hours of the administration nucleic acid vectors and vascular permeabilizing agent); no vasodilating agent is delivered to the blood vessels prior, or during, to the administering of the nucleic acid vectors.
• VPAs Vascular Permeabilizing Agents
• the present invention employs vascular pemerabilizing agents (VPAs) in conjunction with a nucleic acid vector during systemic administration to subject. While the present invention is not limited to any particular VPA, agents that are able to bind Fltl and Flkl are prefened (e.g. VEGF molecules, such as VEGF165 or fragments or variants thereof).
• VPAs in General The present invention is not limited by the type of VPA that is employed.
• VPAs include, but are not limited to, histamine, acetylcholine, adenosine nucleotides, arachidonic acid, bradykinin, cyanide, endothelin, various endotoxins, interleukin-2, ionophore A23187, nitroprusside, various leukotrienes, oxygen radicals, phospholipases, platelet activating factor, protamine, mannitol, sorbitol, serotonin, tumor necrosis factor, vascular endothelial growth factor (VEGF), empty adeno-associated virus capsids (e.g.
• VPA capsids that do not contain viral nucleic acid
• venoms vasoactive amines
• the VPA of the present invention is non-toxic such that it can be administered to a subject without harming the subject.
• a VPA is considered "non-toxic" if it does not have harmful affects on a subject when administered at a dose (with nucleic acid vectors) such that the nucleic acid vectors are able to transduce extravascular tissue at a therapeutic level when administered systemically.
• VEGF e.g. various isoforms, fragments and variants of VEGF.
• a candidate compound e.g. fragment or variant of the above, or some additional compound
• the candidate compound may be substituted for VEGF in Example 1 below.
• the results can be examined to determine if the VPA allows transduction by a viral vector at significant levels (and to determine if the candidate compound is toxic to the animals).
• fragments or variants of the compounds listed above, or other compounds may be screened for usefulness in the methods and compositions of the present invention.
• VEGF molecules are prefened in the methods and compositions of the present invention.
• a "VEGF molecule" includes any VEGF isoform from any species or any fragment or variant of a VEGF isoform that has the same ability to facilitate extravascular transduction by systemic administration of nucleic acid vectors. Fragments and variants of VEGF isoforms may be constructed using known methods in the art such as directed evolution and site directed mutagenesis, and preferably employ Fltl and Flkl as binding partners to isolate good VEGF molecule candidates. Such molecules may be screened for usefulness in Example 1 (e.g.
• the candidate VEGF molecule can in fact serve as a VEGF molecule (e.g. facilitate extravascular transduction via systemic administration of nucleic acid vectors), h certain embodiments, the VEGF molecules (e.g. VEGF-A-165) are administered with NP-1 (neurophilin-1).
• NP-1 neuroophilin-1
• VEGF family members are also known to be potent vascular permeabilizing agents (Senger et al., Science, 1983, 219:983-5, herein incorporated by reference).
• VEGF-A vascular permeabilizing agent
• VEGF-B vascular endothelial growth factor
• VEGF-C vascular endothelial growth factor
• VEGF-D placental growth factor
• PEF placental growth factor
• VEGF-A is the most well studied member of the family, and exhibits both mitogenic and vascular permeabilization activities.
• VEGF-A is expressed as four isoforms, VEGF121 (see Example 3 below), VEGF 165 (known as VEGF164 in mice), VEGF189 and VEGF206, which arise by differential splicing (Houck et al, Mol. Endocrinol., 1991, 5:1806-14). Both VEGF121 and VEGF 165 have been reported to display vascular permeabilization activity. Naturally occurring family members of VEGF are soluble, glycosylated proteins that homodimerize either non-covalently, or via disulfide bonds. Three known receptors have been described, VEGFR-1 (Fltl), VEGFR-2 (Flk-1 or KDR) and VEGFR-3 (Flt-4).
• VEGF-A binds to both Fltl and Flkl, while PDF binds only to Fltl.
• Flt-4 binds only VEGF-C and VEGF-D, which can also bind to Flk-1.
• VEGF-B binds to Fltl (reviewed in Wise et al, J. Biol. Chem., 278:38004-14, 2003, herein incorporated by reference). The interaction between VEGF and Flk-1 has been suggested to be responsible for the induction of vascular permeability (Dvorak et al, J. Histochem. Cytochem., 2001; 49:419-32).
• VEGF 164 proteins that induced vascular permeability
• NP-1 Neuropilin-1
• VEGF- D which binds Flk-1
• VEGF121 has been reported to display vascular permeabilization activity, but lacks the VEGF exon 7 sequences thought to mediate interaction with NP-1.
• VEGF peptides that can induce vascular permeability generally have several common features. These features have been suggested to be an ability to interact with Flk-1 and, possibly via heparin, with NP-1.
• VEGF-A Binding of VEGF-A to Flk-2/KDR is mediated by residues 8-109 of the protein (MuUer et al., PNAS USA, 1997, 94:7192-7), and a fragment containing amino acids 1-110 binds Flk-1 as tightly as does VEGF165 (the human homologue of mouse VEGF164) (Christinger et al., Proteins, 1996, 26:353-7). In particular, it has been shown that residues in a hairpin loop between amino acids 80 and 90 are important for binding of VEGF to Flk-1.
• VEGF 165 is employed (e.g. conjugated or unconjugated to a nucleic acid vector such as AAV6 or AAVl).
• VEGF121 is employed as the VPA in the present invention (e.g. since it shares all of its sequence in common with the N-terminal 121 amino acids of VEGF 165 (Robinson and Stringer, J. Cell. Sci, 2001, 114:853-65).
• a fragment of VEGF121 is employed as the VPA in the present invention (e.g. conjugated or not conjugated to a nucleic acid vector such as AAV). It is noted that this fragment of VEGF121 is a good facilitator of vector delivery to muscle as shown in Example 3 below. hi certain prefened embodiments, fragments from VEGF121 (or any other VEGF molecule) could be used to modify the capsid structure of AAV vectors to render them inherently capable of vascular permeabilization without the need for added soluble VPA such as VEGF.
• Such fragments include, but are not limited to are: VEGF164, VEGF165, VEGF121, VEGFl-110, VEGF 8-109, VEGF8-99, VEGF25-108, VEGF50-108, VEGF50- 99, VEGF 70-108, VEGF 70-99, VEGF75-108, VEGF75-99 or any fragment similar to these fragments.
• Another protein that could be incorporated into a viral capsid is Substance P, which is only 11 amino acids in length (See, Chang et al., P. Nat. New Biol. 232:86-87,
• VEGF vascular endothelial growth factor
• VEGF dimers are readily obtained when VEGF monomers are produced in bacteria, yeast, and 293 cells (Ma, et al., 2001, Biomed Environ Sci 14:302-311; and Mohanraj et al., 1995, Growth Factors 12:17-27).
• a number of approaches could be employed to enable dimer formation on the capsid.
• a linker is one of the short loops that connect the coiled-coil units of the dystrophin spectrin- like repeats (Koenig, 1990, J Biol Chem 265:4560-4566, herein incorporated by reference).
• VPAs empty adeno-associated viral capsides
• AAVs that do not contain viral nucleic acid
• the empty AAV capsids are AAV6 capsids.
• Example 5 below describes methods of generating different populations of AAV6 capsids (e.g. with different percentages of empty vs. nucleic acid containing capsids).
• compositions comprising less than 1% of empty capsids are employed to enhance systemic transduction of vectors (e.g. when intravenously injected).
• compositions comprising at least 50%, or at least 95% or at least 99% empty capsids are employed to enhance systemic transduction of vectors.
• VPAs such as those described above
• VPAs are combined with the empty capsids.
• only fragments of the AAV capsids are employed to enhance systemic tranduction.
• Additional potential VPAs include, but are not limited to, placental growth factor (PIGF), platelet-derived growth factor (PDGF), VEGF-C, heterodimeric VEFG-
• PIGF placental growth factor
• PDGF platelet-derived growth factor
• VEGF-C heterodimeric VEFG-
• nucleic Acid Vectors contemplates the use of nucleic acid vectors with the compositions and methods of the present invention. The present invention is not limited by the choice of nucleic acid vector employed. In general, nucleic acid vectors suitable for use with the methods and compositions of the present invention, for example, should be able to adequately package and cany a nucleic acid sequence of interest (as well as sequences necessary for expression of the nucleic acid sequence in a cell).
• a number of suitable vectors are known in the art including, but are not limited to, the following: A) Adeno- Associated Viral Vectors; B) Adenoviral Vectors; C) Second Generation Adenoviral Vectors; D) Gutted Adenoviral Vectors; E) Lentiviral Vectors; and F) Retroviruses.
• B) Adenoviral Vectors Adenoviral Vectors
• C) Second Generation Adenoviral Vectors B
• D Gutted Adenoviral Vectors
• E Lentiviral Vectors
• Retroviruses Retroviruses.
• Those skilled in the art will recognize and appreciate that other vectors are suitable for use with methods and compositions of the present invention. Indeed, the present invention is not intended to be limited to the use of the recited vectors, as such, alternative means for delivering the compositions of the present invention are contemplated.
• compositions of the present invention are associated with retrovirus vectors and herpes virus vectors, plasmids, cosmids, artificial yeast chromosomes, and bacterial artificial chromosomes. Exemplary delivery approaches are discussed below.
• A_deno-Associated Virus Vectors In prefened embodiments, the nucleic acid sequence of interest is inserted in adeno- associated vectors (AAV vectors), such as AAV6 or AAVl.
• AAV Adeno-associated viruses are non-pathogenic members of the parvo virus family. At least six serotypes of AAV have been identified from human cells and tissues, and these are refened to as AAV serotypes 1-6, or simply AAVl -6 (Rutledge et al., J. Virol, 72:309-19, 1998, and Xiao et al, 73:3994-4003, 1999, herein incorporated by reference). Parvo viruses are known to infect many different species, and recently two additional non-human serotypes named AAV7 and 8 have been described (Gao et al., 2002, PNAS USA, 99:11854-9, and WO03052051, herein incorporated by reference).
• AA_V9 Another serotype, AA_V9, is discussed in WO03052052 (herein incorporated by reference). It is also believed that a large number of additional AAVs have been discovered and will be made public in the near future (e.g. by James Wilson at Pennsylvania University, see Gao et al., J Virol. 2004 Jun;78(12):6381-8, herein incorporated by reference). These viruses display a broad host range, and the different serotypes display numerous differences in their tropism for different cell types (e.g. Chao et al, Mol. Ther. 2:619-623, 2000, and Grimm et al, Mol. Ther., 2003, 7:839-50).
• AAV vectors have been exploited by numerous laboratories to develop gene transfer vectors that may be of use in gene therapy applications.
• AAV vectors have a single stranded genome composed of approximately 4,700 bases flanked by short, inverted tenninal repeats (Muzyczka, 1992, 158:97-129, herein incorporated by reference). The genome contains 2 genes, rep and cap, that encode 4 Rep proteins and 3 capsid proteins, VPl, VP2 and VP3.
• VP proteins form the viral capsid.
• the different fonns differ from one another by the length of the N-terminal region, and 60 copies of the capsid protein are present in a viral particle in ratios of VP1:VP2:VP3 of 1:1:8.
• Recombinant AAV vectors can be prepared in the laboratory using a variety of different methods (e.g., Collaco et al, Gene, 1999, 238:397-405, and Xiao et al., J. Virol., 1998, 72:2224-32).
• the rAAV vector genomes contain a gene expression cassette flanked by the terminal repeats (TR) from wild-type AAV.
• the AAV TRs are thus the only portion of wild-type AAV that is packaged into the recombinant AAV vector.
• Such a rAAV can be grown in tissue culture cells, such as human 293 cells, that have been co- transfected with a transfer plasmid and with a helper virus or with helper plasmids (e.g. Grimm et al, Hum. Gene Ther., 1998, 9:2745-60).
• the transfer plasmid is composed of a plasmid vector containing the recombinant AAV genome.
• the helper functions can be provided by infection of the 293 cells with herpes viruses, adenoviruses or by co- fransfection with one or more plasmids canying appropriate genes for helper function.
• rAAV genomes are prepared by a three plasmid co-transfection method: the first plasmid is the terminal-repeat-expression cassette plasmid (transfer plasmid); the second plasmid contains the AAV genes rep and cap, and the third plasmid carries critical helper genes from adenovirus, such as E4, E2, and VARNA (Fenari et al., Nature Med. 1997, 3:1295-1297).
• AAV vectors can be prepared from any of the reported serotypes 1-8 (or other that are discovered) using genomes, rep and cap genes derived from the individual wild-type AAV viruses.
• pseudotyped vectors may be prepared using an AAV2 genome, the rep gene from AAV2, and the cap gene from the particular serotype of interest (e.g., Rabinowitz et al., J. Virol.
• rAAV6 pseudotyped rAAV6
• rAAV vectors are particularly attractive for human gene therapy applications, as they have been reported to efficiently transduce numerous cell types and support stable, long-term gene expression without induction of inflammation, an immune response or other pathogenic responses.
• dystrophin-deficient muscles from the mdx mouse model for Duchenne muscular dystrophy (DMD), or in the sarcoglycan-deficient Bio 14.6 hamster model for limb-girdle muscular dystrophy (LGMD)
• an inflammatory response has been observed following delivery of AAV2 vectors that express an immugenic protein, such as beta-galactosidase, under control of a ubiquitously active promoter, such as the human cytomegalovirus immediate early promoter.
• an immugenic protein such as beta-galactosidase
• a ubiquitously active promoter such as the human cytomegalovirus immediate early promoter.
• Other proteins delivered to the dystrophic mdx mouse, such as dystrophin have not elicited an obvious immune response.
• tissue-restricted promoter element such as that derived from the muscle creatine kinase gene, is used and the intracellular localization of the expressed fransgene.
• AAV capsids The tropism of rAAV vectors is derived from the nature of the capsid gene used for growth and encapsidation of the vectors (Grimm et al., 2003 and Rutledge 1998). Several research groups have exploited the tropism of the different capsids to increase the transduction efficiency of several different tissues or cell types. For example, AAVl,
• AAV5 and AAV6 have been shown to display a greater ability to transduce skeletal muscle cells than does AAV2 (e.g. Scott et al., 2002).
• a number of investigators have attempted to modify the natural sequence or structure of either individual capsid proteins, or the overall structure of the viral capsid. For example, several groups have attempted to alter rAAV tropism by directly mutating the sequence of an AAV capsid gene. These mutations have taken the form of amino acid substitutions, additions, deletions and insertions (e.g. Buning et al., Gene Ther., 2003, 10:1142-51 and Grifman et al, Mol. Ther. 2001, 3:964-75). These sequence modifications have generally been within the middle of the capsid sequence.
• the rAAV particles grown in the presence of this modified capsid could be packaged and purified at high titer, and enabled tropism modifications by enabling binding of the inserted peptide and specific IgG proteins directed at different epitopes (Ried et al, J. Virol, 2002, 76:4559-66).
• AAV2 capsid mutants were generated by inserting a 14 amino acid peptide into multiple loop structures of the capsid (Girod et al, Nat. Med., 1999, 5:1052-6). These authors reported that all the mutants they isolated supported efficient packaging of rAAV2 vector genomes, although only half expressed the inserted peptide on their outer surface.
• Grifman et al incorporated a variety of peptides up to 13 amino acids in length into AAV2 capsids (Grifman et al, Mol. Ther., 2001, 3:964-75).
• the AAV6 capsid gene encodes 3 capsid proteins that all share a common C-terminal region (see, e.g., U.S. Pat. 6,156,303 to Rutledge et al. describing AAV6; herein incorporated by reference).
• the predicted amino acid sequence of these three proteins, VPl, VP2 and VP3 is shown in U.S. Pat. 6,156,303.
• the longest protein, VPl is 736 amino acids in AAV6, and 735 amino acids in AAV2.
• Recombinant AAV vector capsid proteins can also be modified to the variable region of single-chain antibodies.
• Chimeric AAV capsid genes have also been prepared by co-transfection of DNA fragments of the AAV3 capsid gene together with mutant and non-functional AAV2 capsid genes. Rescued virions all displayed altered tropism, which was shown to have occuned by homologous recombination between the AAV2 and 3 capsid genes, resulting in chimeric capsid genes that incorporated between 16 and 2,200 bases of the AAV3 capsid gene into the recombinant AAV2 capsid gene (Bowles et al, 2003).
• Another approach that attempted to identify sequences in AAVl and AAV2 responsible for the tropism of those serotypes involved transferring discreet regions from AAVl and AAV2 capsid genes to make hybrid
• AAV2/1 capsid vectors that displayed intermediate tropisms for muscle compared with the parental vectors (Hauck and Xiao, 2003). These studies also suggest that rAAV vectors can be grown in cell lines that express two or more different capsid genes derived from multiple different serotypes of AAV. Such recombinant AAV vectors are expected to display different cell binding characteristics than rAAV vectors prepared using a capsid gene from a single serotype.
• Another approach to altering AAV tropism involves modifying the AAV2 capsid gene to encode a particular epitope.
• rAAV vectors prepared in the presence of this modified capsid protein could then be combined in vitro with an immunoglobulin, or antibody, that recognizes or binds to the epitope.
• an immunoglobulin, or antibody that recognizes or binds to the epitope.
• the antibody is a bi-specific single-chain antibody, such as those described by Haisma et al (Cancer Gene Ther., 200, 7:901-4)
• a second ligand can be attached to the antibody to increase the tropism of the rAAV-antibody complex for specific cell types by virtue of their increased binding affinity for receptors on the surface of those cells.
• Methods similar to the above, in some embodiments, could be used such that rAAV particles are re-targeted to bind VEGF receptor proteins Fltl or Flkl (e.g.
• rAAV capsid conjugated to a VPA such as VEGF see below.
• At least one group has incorporated a seven amino acid peptide into the capsid of rAAV2 at position 587, and the resulting modified vectors displayed enhanced targeting of human vascular endothelial cells (Nicklin et al, Mol Ther. 2001, 4:174-81).
• These examples have generally used a fully mutated capsid gene for generation of rAAV particles, such that all the capsid proteins incorporated into the capsid contain the same modification.
• rAAV particles by co-transfecting the vector producing 293 cell line with a mixture of wild-type and mutated capsid genes such that the rAAV particles would contain only a subset of modified capsid proteins, as the AAV particle contains 60 copies of the capsid protein.
• a rAAV vector produced in such a manner would retain tropism characteristics that retain the natural tropism of the wild-type AAV serotype, for example for striated muscle, while also allowing increased binding to additional cell types or receptors, such as a receptor present on vascular endothelial cells.
• a rAAV vector were prepared in the presence of the AAV6 capsid gene and a mutant AAV6 capsid that incorporated a ligand for the VEGF receptor proteins Fltl and Flkl (e.g., Terman et al, Growth Factors, 1994, 11:187- 95; and A; Autiero, 2003, 9:936-43, both of which are herein incorporated by reference), then that rAAV vector might display an increased ability to traverse vascular endothelial cell barriers in capillaries and transduce striated muscle cells.
• a rAAV vector were prepared in the presence of the AAV6 capsid gene and a mutant AAV6 capsid that incorporated a ligand for the VEGF receptor proteins Fltl and Flkl (e.g., Terman et al, Growth Factors, 1994, 11:187- 95; and A; Autiero, 2003, 9:936-43, both of which are herein incorporated by reference).
• Adenoviral Vectors Self-propagating adenovirus (Ad) vectors have been extensively utilized to deliver foreign genes to a great variety of cell types in vitro and in vivo. "Self- propagating viruses" are those which can be produced by fransfection of a single piece of DNA (the recombinant viral genome) into a single packaging cell line to produce infectious virus; self- propagating viruses do not require the use of helper virus for propagation.
• adenoviral vectors have limitations on the amount of heterologous nucleic acid they are capable of delivering to cells. For example, the capacity of adenovirus is approximately 8-10 kb, the capacity of adeno-associated virus is approximately 4.8 kb, and the capacity of lentivirus is approximately 8.9 kb.
• Second Generation Ad vectors In an effort to address the viral replication problems associated with first generation Ad vectors, so called “second generation” Ad vectors have been developed. Second generation Ad vectors delete the early regions of the Ad genome (E2A, E2B, and E4). Highly modified second generation Ad vectors are less likely to generate replication- ( competent virus during large-scale vector preparation, and complete inhabitation of Ad genome replication should abolish late gene replication. Host immune response against late viral proteins is thus reduced [See Amalfitano et al, "Production and Characterization of Improved Adenovirus Vectors With the El , E2b, and E3 Genes Deleted," J. Virol. 72:926- 933 (1998)].
• E2A, E2B, and E4 genes from the Ad genome also provide increased cloning capacity.
• the deletion of two or more of these genes from the Ad genome allows for example, the delivery of full length cDNA dystrophin genes, or mini- dystrophin genes via Ad vectors [Kumar-Singh et al, Hum. Mol. Genet., 5:913 (1996)].
• Gutted vectors are defective viruses produced by replication in the presence of a helper virus, which provides all of the necessary viral proteins in trans. Since gutted vectors do not contain any viral genes, expression of viral proteins is not possible. Recent developments have advanced the field of gutted vector production [See Hardy et al, "Construction of Adenovirus Vectors Through Cre-lox Recombination,” J. Virol. 71:1842- 1849 (1997) and Hartigan-O'Conner et al, "Improved Production of Gutted Adenovirus in Cells Expressing Adenovirus Preterminal Protein and DNA Polymerase,” J. Virol. 73:7835-7841 (1999)].
• Gutted Ad vectors are able to maximally accommodate up to about 37 kb of exogenous DNA, however, 28-30 kb is more typical.
• a gutted Ad vector can accommodate the full length dystrophin or cDNA, but also expression cassettes or modulator proteins.
• Lentiviral Vectors Vectors based on human or feline lentiviruses have emerged as another vector useful for gene therapy applications. Lentivirus-based vectors infect nondividing cells as part of their normal life cycles, and are produced by expression of a package-able vector construct in a cell line that expresses viral proteins. The small size of lentiviral particles constrains the amount of exogenous DJ A they are able to cany to about 10 kb.
• Retroviruses Vectors based on Moloney murine leukemia viruses (MMLV) and other retroviruses have emerged as useful for gene therapy applications. These vectors stably transduce actively dividing cells as part of their normal life cycles, and integrate into host cell chromosomes. Retroviruses may be employed with the compositions of the present invention (e.g. gene therapy), for example, in the context of infection and transduction of muscle precursor cells such as myoblasts, satellite cells, or other muscle stem cells.
• MMLV Moloney murine leukemia viruses
• AAV particles have bee reported to be between 20-25 ⁇ m in diameter as determined by electron microscopy of purified viral particles (Xie et al, Acta. Crystallogr. D. Bioll Crystallogr., 2003, 59:959-70).
• Adenovirus particles are icosahedral shaped and have a diameter between 70-100 nrn.
• the data generated during the development of the present invention indicates that rAAV vectors are readily transfened to striated muscle via the vasculature using the systemic administration methods described herein, whereas adenoviral vectors do not accumulate to a large degree in muscle with this approach. Consequently, viral vectors around the size of AAV (e.g.
• vectors with a diameter around lOmn to 50nm or 5nm to 70nm are the prefened vectors for gene delivery to muscle via the vasculature using the methods of the present invention.
• Other vectors with diameters in the range of 70-100 run may also be transferred with these methods, although they may be less efficient (e.g. a higher dose may be required).
• nucleic acid vectors of the present invention are conjugated to a VPA or to a VPA receptor/ligand.
• the present invention is not limited by the method used to generate the nucleic acid vector conjugates.
• one nucleic acid vector, the AAV may be mutated to allow for increased vascular permeabilization and subsequent penetration of the endothelial cell barriers lining capillaries.
• peptides derived from a VEGF molecule e.g.
• a VEGF isoform are directly incorporated into the AAV capsid by inserting a nucleic acid sequence encoding these peptides into the AAV capsid gene.
• These peptides may be a fragment (or variant) of a VEGF isoform up to the full size of the VEGF isoform.
• Prefened fragments of a VEGF isoform may range in size from 25 to 100 amino acids (see above) (e.g. Yano et al, Methods Mol. Med., 2003, 74:391-8, herein incorporated by referene).
• a similar method could be applied in which peptides from a number of other vascular permeabilizing proteins, such as placental growth factor (Autiero, et al, 2003, Tjwa, et al, 2003), are incorporated into the AAV capsid (e.g. using the capsid-peptide conjugation methods known in the art and described above in part II).
• placental growth factor e.g. placental growth factor
• the full-length, or various fragments from a VEGF isoform are appended onto the C-terminal end of the VP3 protein. This approach may be accomplished by cloning a nucleic acid sequence encoding the appropriate VEGF amino acid sequences onto the 3' end of the VP3 gene in an AAV helper plasmid.
• a similar method may also be used in which peptides from other vascular permeabilizing proteins, such as placental growth factor (PGF, also known as PLGF), are incorporated into the AAV capsid at the carboxy-terminal end of VP3.
• PPF placental growth factor
• a single-chain variable region fragment e.g. from a monoclonal antibody or Fab
• a vascular penneabilizing agent such as VEGF 164 or VEGF165
• This approach may be accomplished by cloning a nucleic acid sequence encoding the appropriate single-chain variable region fragment onto the 3' end of the VP3 gene in an AAV helper plasmid.
• the conesponding vascular permeabilizing agent such as VEGF164 or VEGF165
• VEGF164 or VEGF165 is then be attached to rAAV particles grown in the presence of this modified capsid protein, to facilitate binding of the rAAV particle to the vascular permeabilizing agent in vitro.
• a single-chain variable region fragment e.g. from a monoclonal antibody or Fab
• a receptor for vascular permeabilizing agent such as one of the VEGF receptors Fltl and Flkl
• an AAV capsid gene in an AAV helper plasmid is modified by insertion of a nucleic acid sequence encoding an immunoglobulin-binding domain derived from protein A.
• IgG molecules specific for one or more vascular permeabilizing agents are attached to rAAV particles grown in the presence of this modified capsid protein, via the protein A motif in the capsid, to facilitate binding of the rAAV particle to the vascular permeabilizing agent(s) in vitro.
• vascular permeabilizing agent e.g. the VEGF receptors Fltl and Flkl .
• a sequence encoding a particular epitope is incorporated into an AAV capsid gene of an AAV helper plasmid.
• rAAV particles grown in the presence of this modified capsid protein are combined in vitro with an immunoglobulin, or antibody, that recognizes or binds to the incorporated epitope.
• an immunoglobulin, or antibody that recognizes or binds to the incorporated epitope.
• a bi-specific single-chain Fv (scFv) antibody that binds to the epitope as well as a vascular permeabilizing agent, such as VEGF 165
• the vascular permeabilizing agent is then be attached to the antibody to generate a rAAV particle physically attached to the vascular permeabilizing agent.
• a modified rAAV particle is expected to display an increased tropism for cells that express the receptor for the particular vascular permeabilizing agent, e.g. the VEGF receptors Fltl and Flkl.
• a bi-specific single-chain antibody composed of an anti-epitope single-chain Fv antibody fused to an scFv antibody directed against either VEGF, or the VEGF receptor.
• a bi-specific scFv antibody composed of an anti-AAV capsid scFv antibody fused to an scFv antibody directed against either VEGF, or the VEGF receptor.
• a purified, rAAV is biotinylated in vivo using the endogenous biotin ligase known to exist in human 293 cells.
• biotinylation simply requires that a short biotin acceptor peptide be inserted into the capsid protein, at any of the various sites described in the above paragraphs, such as amino acid 587.
• the biotinylated rAAV capsid could then be combined in vitro with VEGF, or another vascular permeabilizing agent, conjugated directly to streptavidin or avidin, which display a high affinity for biotin.
• VEGF or another vascular permeabilizing agent
• Such a modified rAAV particle are exipected to display an increased tropism for cells that express the receptor for the particular vascular permeabilizing agent, e.g. the VEGF receptors Fltl and Flkl.
• the biotinylated AAV particle could be conjugated to biotinylated antibodies against the VEGF-receptor, or against VEGF, using avidin as a linker.
• the nucleic acid vectors of the present invention comprise a nucleic acid sequence of interest.
• the nucleic acid sequence of interest is a therapeutic nucleic acid sequence (e.g. when the nucleic acid vector delivers the nucleic acid sequence of interest to the cells of a subject, the nucleic acid sequence of interest is expressed and provides a therapeutic benefit to the subject).
• nucleic acid sequences that encode a protein that is defective or missing in a recipient subject, or a heterologous gene that encodes a protein having a desired biological or therapeutic effect are suitable nucleic acid sequences of interest.
• nucleic acid sequences of interest include, but are not limited to, those encoding proteins used for the treatment of endocrine, metaloic, hematologic, cardiovascular, neurologic, musculoskeletal, urologic, pulmonary, and immune disorders, including such disorders as inflammatory diseases, autoimmune disease, chronic and infectious diseases, such as AIDS, cancer, hypercholestemia, insulin disorders such as diabetes, growth disorders, various blood disorders including various enemias, thalassemias, and hemophilia; genetic defects such as cystic fibrosis, Gaucher's disease, Hurler's disease, adenosine deaminase (ADA) deficiency, aging related symptoms (e.g.
• the nucleic acid sequence of interest encodes a protein that inhibits myostatin (GDF-8).
• Myostatin has been identified as a potent negative regulator of muscle fiber size during myogenesis and after birth (see, McPhenon, Nature. 1997 387:83-
• the biologically active component of myostatin is a protein homodimer that targets membrane-bound activin receptors in skeletal muscle.
• the availability of biologically active myostatin is regulated by a) synthesis of the precursor protein, b) cleavage and dissociation of the myostatin pro-peptide to liberate the active, ligand-binding molecule, c) interaction between the active dimer and other proteins that negatively regulate its activity such as follistatin, Follistatin Related Gene (FLRG), and Growth and Differentiation Associated Serum Protein - 1 (GASP-1) (see, Lee et al, Annu Rev Cell Dev Biol 2004;20:61-86, herein incorporated by reference), and d) negative feedback of signal transduction elements stimulated by myostatin.
• FLRG Follistatin Related Gene
• GASP-1 Growth and Differentiation Associated Serum Protein - 1
• the present invention provides methods of systemic delivery of vectors to a subject such that myostatin inhibitors are expressed.
• the subject has a muscle wasting disorder, such as in the muscular dystrophies, aging, or cancer cachexia, that is treated by this approach (e.g. at least some of the symptoms associated with the muscle wasting are reduced or eliminated).
• the subject is a member of the anned forces or an astronaut who might not exhibit symptoms of pathology, but who could benefit from treatment with the compositions of the present invention.
• the nucleic acid sequence of interest may not have therapeutic value.
• the nucleic acid sequence of interest may be a reporter gene, or a gene used to increase muscle mass (e.g. in a farm animal or human athlete) or the nucleic acid sequence of interest improves physical appearance or alters physical appearance in an animal (e.g. a cosmetic type effect results).
• the nucleic acid sequence of interest in some embodiments, will code for a protein antigen.
• the antigen may include a native protein or protein fragment, or a synthetic protein or protein fragment or peptide.
• antigens include, but are not limited to, those that are capable of eliciting an immune response against viral or bacterial hepatitis, influenza, diphtheria, tetanus, pertussis, measles, mumps, rubella, polio, pneumococcus, herpes, respiratory syncytial virus, hemopriilus influenza type b, chlamydia, varicella-zoster virus or rabies.
• the nucleic acid sequence of interest may also be a normal muscle gene that is effected in a muscle disease (e.g.
• the nucleic acid may be a heterologous gene encoding the full length dystrophin gene (or cDNA sequence), BMD-minigene, AH2-R19 minigene, Laminin-a2, utrophin, a- sarcoglycan, and emerin.
• BMD mini-gene refers to dystrophin cDNAs containing internal truncations .
• Prefened nucleic acid sequences of interest are dystrophin, preferably micro (mini) dystrophin constructs.
• Prefened mini-dystrophin constructs have 4, 8, or 16 spectrin like repeats (e.g. utrophin or dystrophin derived repeats).
• Prefened constructs are provided in WO0229056 and Harper et al, Nature Med., 8:253-261, 2002, both of which are explicitly incorporated by reference as if fully reproduced herein. These references describe constructs with a perfect number of spectrin like repeats (e.g. 4.0 repeats, or 8.0 repeats), as well as constructs that only have a limited number of the four natural hinge regions in the dystrophin gene.
• dystrophins of reduced size are as follows: Yausa et al. FEBS Letters 425:329-336, 1998, which describes a series of truncated dystrophin cDNAs containing 3, 2, 1, or 0 rod repeats (spectrin-like repeats; see figure 1 of this reference); Wang et al. PNAS, 97(25): 13714-13719, December 5, 2000 which describes a series of minidystrophin genes (e.g. with 5 and 6 rod repeats as shown in figure 1; see also WO0183695 to Xiao Xiao, published November 8; Phelps et al, Hum. Mol.
• the microdysfrophm construct ⁇ R -23 / ⁇ CT, contains the actin binding amino-terminal domain, spectrin-like repeats 1, 2, 3, and 24, hinge domains 1, 2, and 4, as well as the WW and cysteine rich domains (designated by the shaded oval labeled, "WW/CR").
• the microutrophin construct, ⁇ U- ⁇ R t-21 / ⁇ CT contains analogous regions to those present in construct ⁇ ⁇ / ⁇ CT with the exception of approximately 50 amino acids after the ZZ domain portion of the cysteine rich domain, designated in Figure 12 as a shaded rectangle labeled "E". The amino acids contained in region "E" are not known to be required for binding to the dystroglycan complex.
• UD-Chimerafrophin/ ⁇ CT-II and DU-Chimeratrophin are hybrid proteins.
• Chimeratrophin/ ⁇ CT-II combines domains from the amino terminal domain through hinge 2 of ⁇ U- ⁇ R 4-21 / ⁇ CT-II and the WW/CR domain of ⁇ R 4-23 / ⁇ CT.
• DU-Chimeratrophin combines domains from the amino terminal domain through hinge 4 of &R 4 - 23 / ⁇ T and the WW/CR domain from ⁇ U- ⁇ R ⁇ / ⁇ CT-II.
• the sequence of three micro-utrophin sequences are shown in Figures 13A (SEQ ID NO:l), 13B (SEQ ID NO:2), and Figure 13C (SEQ ID NO:6). Changes to these sequences could also be made and tested (e.g. substituted for the micro-dystrophins described in the Examples below). In this regard, additional microutrophin sequences could be identified and used to treat subjects with muscle wasting diseases.
• the term "dystrophin-like" nucleic acid refers to any nucleic acid that encodes a protein that could be used to functionally substitute for the dystrophin protein, including those that are able to link the actin cytoskeleton to dystrogylcan, or the actin cytoskeletal to the extracellular matrix (e.g. cDNA for CT GalNAc transferase, see, Nguyen et al, PNAS, 2002, 99:5616-21, herein incorporated by reference). Examples include, but are not limited to, micro-dystrophins, utrophin, micro-utrophins, dystrophin/utrophin chimeras, and micro-dystrophin/utrophin chimeras.
• the nucleic acid sequence of interest is Igf-1 (e.g. any isoform from any species).
• nucleic acid sequences encoding Igf-1 are the mouse sequence (NM_010512) and the rat sequence (NM_178866). Additional examples include murine Igf-I Ea which contains exons 1,3,4,6 (SEQ ID NO:4), murine Igf-I Eb which contains exons 1,3,4,5,6 (SEQ ID NO:3), and human Igf-1 (SEQ ID NO:5).
• Vectors encoding Igf-1 maybe administered to subject to treat muscle wasting disorders and particularly to treat muscle wasting associated with aging (e.g. treat a human 65 or older, 70 or older or 75 or older).
• Igf-1 methods for systemic delivery if Igf-1 similar to those described in Example 8 (e.g. except used to treat a human with a human form of the Igf-1 sequence) may be employed (e.g. to treat an elderly human).
• the Igf-1 leads to over-expression of Igf-1 in the subject (e.g. 2 times, 10 times, or 30 times or 50 times the natural level of Igf- 1 ) .
• the present invention provides methods of treating a subject with both Igf-1 and a dystrophin type nucleic acid (full or micro-dystrophin, utrophin, micro-utrophin, dystroph-utrophin chimeric, or any other sequence that can effectively substitute for dystrophin) such that both Igf-1 and the dystrophin type protein are expressed in a subject.
• a dystrophin type nucleic acid full or micro-dystrophin, utrophin, micro-utrophin, dystroph-utrophin chimeric, or any other sequence that can effectively substitute for dystrophin
• kits and compositions comprising vectors with both Igf-1 and dystrophin like sequence (e.g. in the same vector or different vectors; or in the same composition or separate compositions).
• Nucleic acid sequences of interest may also be antisense molecules (e.g. for blocking the expression of an abnormal muscle gene).
• the nucleic acid sequence may also code for proteins that circulate in mammalian blood or lymphatic systems. Examples of circulating proteins include, but are not limited to, insulin, peptide honnones, hemoglobin, growth factors, liver enzymes, clotting factors and enzymes, complement factors, cytokines, tissue necrosis factor and erythropoietin.
• Nucleic acid sequenes of interest may also genes encoding proteins that are to be produced in muscle cells in vitro or in vivo.
• the nucleic acid sequence of interest is an siRNA (RNAi) molecule that is able to suppress a targeted RNA transcript.
• RNAi represents an evolutionary conserved cellular defense for controlling the expression of foreign genes in most eukaryotes, including humans. RNAi is triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of single-stranded target RNAs homologous in response to dsRNA.
• siRNAs small interfering RNA duplexes
• siRNAs are generally approximately twenty-one nucleotides in length (e.g. 21-23 nucleotides in length), and have a base-paired structure characterized by two nucleotide 3'-overhangs.
• RISC RNA- induced silencing complex
• siRNAs have become powerful reagents for genome-wide analysis of mammalian gene function in cultured somatic cells. Beyond their value for validation of gene function, siRNAs also hold great potential as gene-specific therapeutic agents (Tuschl and Borkhardt, Molecular Intervent. 2002; 2(3):158-67, herein incorporated by reference). The fransfection of siRNAs into animal cells results in the potent, long-lasting post- transcriptional silencing of specific genes (Caplen et al, Proc Natl Acad Sci U.S.A.
• siRNAs are extraordinarily effective at lowering the amounts of targeted RNA, and by extension proteins, frequently to undetectable levels.
• the silencing effect can last several months, and is extraordinarily specific, because one nucleotide mismatch between the target RNA and the central region of the siRNA is frequently sufficient to prevent silencing Brummelkamp et al, Science 2002; 296:550—3; and Holen et al, Nucleic Acids Res. 2002; 30: 1757-66, both of which are herein incorporated by reference.
• the nucleic acid sequences of interest are operably linked to a tissue-specific promoter and/or enhancers. In this regard, expression of the nucleic acid sequence of interest can be primarily localized to one target tissue type.
• the prefened route of administration of the compositions of the present invention is systemic administration to a blood vessel of a subject without one or more systemic administration aids.
• the present invention is not limited to un- aided systemic administration or to only systemic administration.
• the compositions of the present invention e.g.
• nucleic acid vector with a VPA, or high doses of AAV6) are administered to a subject via a route selected from: intravenously, intra-muscularly, subcutaneously, intradermally, intraperitoneally, intrapleurally, intrathecally, orally, rectally or topically.
• formulations for such administrations may comprise sterile water or physiological saline.
• VEGF vascular endothelial growth factor
• VPAs described above including VEGF and empty AAV6 capsids
• pre-injection vectors with a sequence are then injected into the subject (e.g. within 30 seconds, 2 minutes, or 10 minutes).
• the time per single infusion employed with the methods of the present invention varies from 3 second to 60 minutes (e.g. 5 second, 45 second, 10 minutes or 30 minutes).
• systemic administration of the compositions of the present invention can be to the veins or arteries or to both.
• various catheters can be employed to access various locations in a subject.
• the maximal dosage of AAV6 in solution is about 10 15 particles per ml.
• the VPA is initially administered with the vector (e.g. in the same composition or separate compositions administered close in time), and then a second administration occurs later (e.g. 1 min, 5 min, 15 min, 1 hour or 3 hours later) that contain only the VPA and not additional vector.
• vector administration is repeated a number of times, while avoiding or minimizing an immune response from a subject, h particular embodiments, the vector is administered to the subject over a period of 5-1O days. Wlender not limited to any mechanism, it is believed that human immunity to the vectors (e.g. AAVs) takes about 5-10 days to develop, allowing repeat administration in this time window. In other embodiments, transient immune suppression is utilized to prevent an immune response (e.g. a humoral immune response to viral capsid proteins).
• an immune response e.g. a humoral immune response to viral capsid proteins
• EXAMPLE 1 Adeno-associated viral transduction with VEGF This example describes systemic administration of an adeno-associated virus in combination with VEGF to animals without the use of systemic administration aids. This example also describes systemic administration of AAV6 without a vascular penneabilizing agent and without the use of systemic administration aids.
• Virus Production Recombinant adeno-associated virus(rAAV)vectors containing the various promoter and fransgene casettes (see below) flanked by AAV serotype 2 terminal repeats were packaged using the Rep and Cap open reading frames of AAV serotype 6.
• rAAV adeno-associated virus
• 293D cells were transfected at a density of 3.5 to 4.0 x 10 6 cells per 10cm diameter tissue culture dish. Transfection was carried out via calcium phosphate precipitation. Each 10cm diameter plate was transfected with 20 ⁇ g of the packaging plasmid, pDG6, and lO ⁇ g of the appropriate vector genome containing plasmid. Medium was exchanged to serum free medium 16 to 24 hours post transfection and 72 hours post transfection cells and medium were collected.
• the cells and medium were reduced to a cleared lysate via passage through a microfluidizer (Microfluidics, Newton, MA., model Ml 10S) and loaded onto a HiTrap Heparin column (Amersham, NJ.) using an AKTApurifier 10 HPLC (Amersham).
• Vectors were eluted from the column in 400mM NaCl supplemented Ringer's solution and dialyzed to Ringer's.
• Animal strains used were either the wild-type, C57B1/10J, or dystrophic, C57Bl/10ScSn-Dw_ -r ⁇ c_x;/J.
• mice Young adult (6-8 wk) mice were injected with a range of rAAV ⁇ 2.5xl0 10 to ⁇ lxl0 13 vector genomes in 250 ⁇ l via tail vein while conscious. Mice were administered 0-20 ⁇ g of recombinant human VEGF- 165 (R & D Systems, MN) in 50 ⁇ l of Ringer's solution containing 0.05% mouse serum albumin and 2 IU heparin either as i.v. pre-treatment 10 min prior to administration of vector, or as i.v. co-treatment with vector.
• FIG. 1 shows a plate demonstrating the gray/black muscles of mice receiving vector and VEGF/heparin, as well as mice receiving vector alone.
• the dark shading is a reaction generated from the enzyme produced by the gene (LacZ) delivered by the virus, h particular, figure 1 demonstrates transduction of cardiac, trunk, and limb muscles of mice following systemic delivery of an AAV6-CMV-LacZ delivered via a single I.V. injection into the tail vein of adult animals. Note the differences in intensity of staining between rows 2 and 3 in figure 1, indicating the greatly increased transduction efficiency with co- treatment of VEGF.
• FIG. 2 shows a graph demonstrating the enzymatic activity measured in muscles and various organs from mice receiving various doses of vector with and without co- treatment of VEGF and heparin.
• figure 2 shows /3-galactosidase activity (driven by the CMV promoter) in various muscles and organs following systemic delivery of 2 x 10 11 or 10 12 doses of viral vector with and without VEGF.
• Virus was injected into young adult mice (6 to 8 weeks) while conscious as a bolus in a volume of ⁇ 300 ⁇ l into the tail vein. Animals were sacrificed approximately 10.5 days post injection.
• Muscles and organs were isolated and homogenized. The extract was used in a luminescent assay for ⁇ - galactosidase activity. If bars are absent it indicates a level of activity not detectable above background. Data was collected for the following tissue types: masseter, FDP/FCR (flexor digitorum brevis/ flexor carpi radialis), bicep, tricep, heart, diamprham, quadricep, gastocnemius, masseter, soleus, tibialis anterior, eye, brain, lung, intestines, liver, kidney, spleen, and testes.
• FDP/FCR flexor digitorum brevis/ flexor carpi radialis
• FIG. 2 shows that, with the exception of soleus limb muscle, no other tissue from 2xl0 ⁇ does mice without VEGF failed to register above contr ls.
• this figure demonstrates that there is considerable differences in the level of transduction achieved at the low dose (2xlO n ) with VEGF compared to without VEGF (i.e. VEGF made a big difference in transduction efficiency at 2x10 11 as only soleus limb muscle was above background at this level without the presence of VEGF).
• FIG. 3 shows various muscles of the body transduced with / (3-galactosidase following a single i.v. injection (using the AAV6-CMV-LacZ constructs into a conscious animal). The gray staining is indicative of positive signal. 1 x 10 12 vector genome and 2 IU of heparin were delivered into a conscious mouse via the tail vein in a bolus of ⁇ 300 ⁇ l The mice were sacrificed approximately 11 days post injection.
• FIG 3 shows that the various muscles exhibit approximately 90% or above transduction on the basis of individual muscle fibers which stain positive for /3-galactosidase activity. These results compare favorably or exceed reported values in the literature using intramuscular injections (which was the most effective method to date, although limited in the amount of muscular tissue transduced). Similar results were seen when the virus and heparin were administered with VEGF though this vasodilator and permeability factor was not essential for widespread transduction at this viral dose.
• Figure 4 shows a transverse section of a mouse heart harvested l id after a single systemic delivery of lxlO 12 viral genomes (i.e.
• AAV6-CK6-LacZ This section describes the results of experiments with an AAV6 containing the lacZ gene with the muscle specific creatine kinase promoter driving expression of the -gal reporter gene (AAV6-CK6-LacZ).
• Various muscles of the body (mouse body) were transduced with ⁇ -galactosidase following a single i.v. injection of AAV6-CK6-LacZ.
• the results of this administration were seen after various muscle groups were isolated from the sacrificed mice. The blue staining in these muscle groups (not shown) was indicative of 19 1 ⁇ positive signal.
• AAV6-CK6-micro-dystrophin This section describes the results of experiments with an AAV6 containing a micro- dystrophin gene with the muscle specific CK6 promoter (AAV6-CK6-micro-dystrophin).
• the micro (or "mini") dystrophin used in this example ( ⁇ R4-R23- ⁇ 71-78) is the same construct shown in Figure 5b in Harper et al, Nature Med., 8:253-261, 2002 (herein incorporated by reference).
• Other micro-dystrophin constructs shown in this paper e.g. Figure la
• WO0229056 herein incorporated by reference
• Figure 27 of WO0229056 also shows the micro-dystrophin construct used in this example (i.e. it shows ⁇ R4-R23- ⁇ 71-78).
• Figure 5 demonstrates a functional conection of dystrophic muscle (tibialis anterior) following a single systemic administration of lxlO 12 viral genomes of pseudotyped AAV6 CK6-micro-dystrophin. Virus was delivered with lO ⁇ g of V.P.F. and 2 LU. of heparin in Ringer's supplemented with .08% mouse serum albumin. Animals were injected at 6-8 weeks of age and sacrificed at 8 weeks post injection.
• the two animal strains used were the wild-type C57B1/10J and the dystrophic C57B1/10ScSn-D . - _&/J. i
• the assay with the mice in this example was a contraction-induced injury type assay. It is known that "stretches" do considerable damage to dystrophic muscle, and dystrophin has been shown to protect muscle fibers from damage from stretches (known officially as eccentric or lengthening contractions). This procedure is described in Delloruso et al, J. Muscle Res. and Cell Motility, 22:467-475, 2001, herein incorporated by reference. Briefly, in this example, the muscles of mice were administered a single stretch whilst maximally stimulated (i.e.
• LCI in figure 5 is the maximum force generated by the muscle 10s after it has endured a single contraction induced injury.
• LC2 in figure 5 is the maximum force generated by the muscle 10s after it has been subjected to the second contraction induced injury, "lmin” in figure 5 is the maximum force produced by the muscle when recorded 60s after the third stretch has been applied. Values in the graph have been presented, relative to the initial force output of respective muscles, so the smaller the bar, the more severe the deterioration in force production experienced as a result of the stretch protocol.
• t statistic is given on the graph for a one tailed t-test and refers to the difference between mdx and treated mdx (Tmdx) mice.
• This data represents a partial protection from contraction induced injury following a systemic delivery on par with the best results to date from a direct infra-muscular injection of virus.
• Figures 6 and 7 demonstrate high levels of mini-dystrophin expression in various muscle groups in mice following a single intravenous injection of AAV6-CK6-rnini- dystrophin with VEGF.
• lxlO 13 viral genomes of pseudo-typed AA.V6-CK6- micro-dystrophin were delivered in a 300 ⁇ l bolus with lO ⁇ g of V.P.F. (VEGF) and 2 LU. of heparin in Ringer's supplemented with .08% mouse serum albumin.
• the bolus was delivered via the tail vein.
• Figures 6 and 7 above demonstrate that dystrophic animals have extremely widespread expression of approximately wild-type levels of micro-dystrophin in both the quadriceps (Quad) muscle (fig. 6), as well as in the tibialis anterior (T.A., fig.
• This specific vector did not elicit significant dystrophin expression in either the heart or the diaphragm, but this is attributable to the activity (or lack thereof) of the CK6 promoter cassette used, within these muscles.
• Other vectors with a constitutive promoter such as CMV demonstrate strong expression in the heart and diaphragm.
• EXAMPLE 2 Systemic AAV Administration to a Large Mammal This Example describes systemic AAV transduction in a dog. In this example, 4 x
• rAAV6/RSV-hpAP 10 13 vg/kg of rAAV6/RSV-hpAP (hpAP is human placental alkaline phosphatase; RSV is a promoter from Rous Sarcoma Virus) was injected into the jugular vein of a 2 month old beagle. Analysis of hpAP expression in the heart and diaphragm occuned at 3 weeks. The results are presented in Figure 8, with the top two panels showing a control and the bottom two panels showing the injected canine tissue. This Example could be repeated with the addition of a vascular permeabilizing agent, such as VEGF-A.
• VEGF-A vascular permeabilizing agent
• EXAMPLE 3 Systemic Transduction with VEGF-A121 This Examples describes systemic transduction of mice with AAV using VEGF-A 121 rather than VEGF-A 165 .
• Miice were injected with 8xl0 12 vg kg of rAAV6/CMV-lacZ +/- 260 ug/kg VEGF-A 121 via the tail vein 11 days prior to analysis of ⁇ gal activity.
• EXAMLPE 4 Intravenous and Intra-Arterial Variable Timing Systemic Vector Delivery This Example describes success with both intravenous and infra-arterial systemic viral vector administration.
• mice were sacrificed, and tibialis anterior, Quadriceps, soleus, and heart muscles were dissected and then sectioned into 10 micron sections.
• FIG 10 there was high level of beta-galactosidase expressions from CMV-lacZ cassettes in heart muscles using a 3 second infusion, as well as with 60 seconds, 300 seconds and with the femoral artery/3 seconds group.
• This Example shows that systemic transduction can be accomplished using multiple different delivery durations.
• EXAMPLE 5 Enhancing Systemic Transduction with Empty AAV6 Capsids
• AAV6 viral capsids i.e. AAV6 particles without associated nucleic acid
• the vector used in this example was rAAV6-CMVlacZ.
• This vector was purified with heparin affinity chromatograph ("column-purified) to generate a preparation with approximately 95% empty AAV6 capsids, or was purified by cesium-chloride (CsCl- banded) to generate a preparation with approximately 50% empty AAV6 capsids.
• Wild • 19 type mice were intravenously injected with 1 x 10 pseudotype-6 AAV from either: 1) the "column-purified” preparation; 2) the “CsCl-banded” preparation, or 3) the "CsCl-banded” preparation combined with additional empty capsids taken from a preparation similar to the "column purified” preparation.
• Exogenous beta-galactosidase activity (darker gray) in the heart, diaphagrm, and tibialis anterior was examined in cryosection of the mice after 12 days (see Figure 11). This Figure shows greater systemic transduction with the 95% empty capsid preparations ("column purified” preparations), indicating that empty AAV capsids enhances systemic transduction of vectors.
• EXAMPLE 6 Micro-Utrophin Treatment of mdx Mice This Examples describes the treatment of mdx mice with micro-utrophin nucleic acid sequences. Mice were injected with 1 x 10 12 vector genomes (rAAV6:CK6-mico- utrophin, which is SEQ ID NO: 1 , shown in Figure 13 A) and analyzed at 6 weeks post injection, hnmunofluorescence staining of a tibialis anterior muscle of the mdx mouse using an antibody against murine utrophin showed uniform staining, hnmunofluorescence staining with ⁇ -syntropin also shows micro-utrophin restores expression of utrophin and dystrophin associated proteins such as ⁇ -syntrophin. This Example could be repeated with the micro-utrophin sequence shown in Figure 13B (SEQ ID NO:2).
• EXAMPLE 7 Co-Delivery of IGF-1 and Dystrophin This Examples describes the co-delivery of IGF-1 and dystrophin and the synergistic results achieved with such administration. In particular, this Example describes the delivery of IGF-1 and dystrophin to mdx (dystrophic) mice via AAV vectors.
• Igf-I Ea contains exons 1,3,4,6 (see SEQ ID NO:4 in Figure 19), while Igf-I Eb contains exons 1,3,4,5,6 (see SEQ ID NO:3 in Figure 19).
• Exon 5 in Igf-I Eb has an insert of 52 bp, which results in the alternative C-terminus of " the peptide.
• Igf-I Eb is up-regulated in muscle subjected to stretch and therefore also refened to as mechano growth factor (MGF) (Yang et al, Journal of Muscle Research & Cell Motility 17:487-495, and McKoy et al., J Physiol 516 (Pt 2):583-592, herein incorporated by reference).
• MMF mechano growth factor
• Both muscle specific isoforms differ from the liver isoforms by using sequences encoded by exon 1 as a leader peptide in contrast to exon 2.
• the Igf-I Ea cDNA was then cloned into the EcoRI-Hindlll site in the polylinker of pMCS-CMV (Sfratagene, La Jolla, CA) that carried the CMV promoter and a bovine growth hormone polyadenylation site.
• the CMV promoter was removed with Mlul and SacII, and replaced by the muscle-specific CK6 promoter.
• the complete expression cassette was then excised with Notl and moved into a pAAV vector backbone, containing serotype 2 inverted terminal repeats (Sfratagene).
• micro-dystrophin cDNA (DR4-R23/DCT, see Figure 14A) is a truncated version of the full-length dystrophin cDNA and was generated by introducing deletions between repeat 4 and repeat 23 within the rod domain, and a deletion of the C- terminal domain (see Harper et al, Nature Medicine 8:253-261, 2002, herein incorporated by reference).
• the micro-dystrophin cDNA was cloned into the Eagl site of the pDD344 plasmid.
• HEK293 cells were co-transfected with 10 mg recombinant AAV vector and 20 mg helper plasmid pDGM6 (Gregorevic et al, Nat Med. 2004 Aug;10(8):828-34, herein incorporated by reference in its entirety) using the calcium phosphate-DNA precipitation method.
• Viral vector purification was performed as described (Blankinship et al, Mol Ther. 2004 Oct;10(4):671-8, herein incorporated by reference in its entirety), hi brief, transfected cells and medium were homogenized through a microfluidizer (Microfluidics, Newton, MA) and cleared through a 0.22 mm filter.
• Vector particles were subsequently purified by affinity chromatography over a HiTrap heparin column (Amersham, Piscataway, NY) using an AKTApurifier 10 high pressure liquid chromatography (HPLC) machine (Amersham), and dialyzed against physiological Ringer's solution.
• the vector titer was determined by quantitative slot blot analysis, using plasmid standards derived from a Notl or Mscl digest of pAAV-Igf-I or pAAV-mdys, respectively.
• Vector genomes were hybridized with transgene-specific probes labeled with the CDP-Star kit (Amersham, Piscataway, NJ) and visualized with a chemoluminescence imager (GeneGnome, Syngene, Frederick, MD).
• mice were anesthetized with either 2,2,2- tribromoethanol (Sigma, St. Louis, MO) or isoflurane (Abbott Laboratories, Chicago, IL) such that they were non-responsive to tactile stimuli, and the tibialis anterior (TA) hindlimb muscles were surgically exposed by performing a small skin incision parallel to the muscle.
• 2,2,2- tribromoethanol Sigma, St. Louis, MO
• isoflurane Abbott Laboratories, Chicago, IL
• Viral vector (1-2 x 10 10 vg) in 30 ml physiological Ringer's solution was then carefully injected into the muscle via a Hamilton syringe equipped with a 32-gauge needle. After injection, the skin incision was closed with Nexaband surgical grade adhesive (World Precision Instruments, Inc., Sarasota, FL). Controls for all experiments consisted of sham injections with physiological Ringer's solution.
• mice were anesthetized with 2,2,2-tribromoethanol (Sigma, St. Louis, MO) to isolate and attach the distal tendon of the TA muscles to a dual-mode servomotor, and position electrodes adjacent to the motor nerve.
• the optimal muscle length (Lo) and the maximum isometric force were determined for each muscle sample.
• muscles were then maximally stimulated and stretched twice from Lo through 40% of muscle fiber length.
• Maximum isometric force was measured after each lengthening contraction and reported as a percentage of initial maximum isometric force.
• the forces produced after LCI and LC2 were used to model the ability of muscles to resist injury.
• the muscles were excised, weighed and prepared for biochemical and histological analysis.
• RNA and DNA isolation and quantitation For biochemical analysis, muscles were frozen in liquid nitrogen for storage at - 80°C until use. Muscles were homogenized (OMNI 5000, OMNI International) in lysis buffer (RNeasy, Qiagen, Sanita Clarita, CA) and then treated with proteinase K (20 mg/ml) to remove connective tissue and collagen. Residual debris was pelleted and the clear supernatant was loaded on a RNA purification column for further extraction according to the manufacturer's instructions (RNeasy, Qiagen). Before washing and eluting the RNA from the columns, an on-column DNase 1 digestion was performed to ensure complete removal of genomic and residual vector DNA (Qiagen). 500 mg total RNA of each sample was electrophoresed to confirm RNA integrity. One mg total RNA of each sample was reverse transcribed into cDNA. Igf-I mRNA transcripts were then quantified by real-time
• Image processing and quantitative measurements Brightfield and fluorescence images of sections were acquired using a Spot-2 and JVC camera respectively via a Nikon E1000 microscope (Melville, NY). Montage images of entire muscle cross sections were graduated using MONTAGE EXPLORER software (Syncroscopy, Frederick, MD) to control stage position during image acquisition. Image analysis was completed using IMAGEPRO software (Media Cybernetics, Silver Spring, MD) with manual on-screen selection of functions. Montage images of hematoxylin and eosin-phloxine stained sections were overlayed with a mask that randomly chose four 400 mm2 microscopic fields per muscle cross section.
• Igf-I and Dystrophin expression To characterize Igf-I expression in normal and dystrophin-deficient muscle, mRNA levels of the Igf-I Ea and Igf-I Eb isoforms were measured by quantitative PCR. Primer pairs were designed to uniquely detect Igf-I Eb or both, Igf-I Ea and Igf-I Eb. Both isoforms were expressed in wild-type and mdx muscles of 9- 13 month old mice, unlike previous data that suggested that Igf-I Eb is not expressed in mdx muscles (Goldspink, et al, J of Physiology 495: 162-163, 1996).
• rAAV vectors were generated that carried expression cassettes in which the muscle-specific creatine kinase promoter/enhancer (CK) drove either the micro- dystrophin (DR4-R24/DCT) or the muscle-specific Igf-I cDNA (Igf-I-Ea) (see Figure 14A).
• CK creatine kinase promoter/enhancer
• TA muscles of nine month old mdx mice were injected either with each vector separately, or in combination.
• dystrophin expression was analyzed by immunohistochemical staining of muscle cross sections using an antibody against the N- terminal domain of dystrophin.
• AAV-mdys injected and AAV-mdys/Igf-I co-injected muscles revealed widespread distribution of dysfrophin-positive fibers throughout the muscles.
• the percentage of dystrophin expressing fibers averaged 40% of the total cross sectional area (Fig. 14B).
• AAV-Igf-I and untreated control muscles contained only a few revertant, dysfrophin-positive fibers, that accounted for less than 5% of the total cross sectional area (Fig. 14B).
• AAV-Igf-I injected mdx muscles demonstrated 50-100 fold higher expression of Igf-I mRNA than mdx control muscles at four months post-injection, hi contrast, co-injection of AAV-mdys/Igf-I resulted in 200-400 fold higher expression of Igf-I mRNA than in mdx control muscles four months following the injection.
• AAV-mdys/Igf-I were analyzed four months post-injection, and compared with the muscles of mdx and wild-type control animals. Muscles injected with AAV-Igf-I and AAV- mdys/Igf-I demonstrated a significant increase in muscle size and mass relative to mdx and AAV-mdys treated muscles. When muscle mass was normalized to whole body mass, mean values were increased 17% for AAV-Igf-I freated and 19% for AAV-mdys/Igf-I co-treated muscles compared with control mdx muscles (Fig. 15 A).
• AAV-mdys/Igf-I co-treated and to a lower extent in AAV-Igf-I freated, animals; however deficiencies in maximum force per cross sectional area were not corrected by Igf-I treatment. Muscles from control and treated groups were also subjected to lengthening under maximum contraction to analyze muscle susceptibility to mechanical damage. All AAV- mdys treated and AAV-mdys/Igf-I co-treated animals demonstrated a significant protection from contraction-induced injury (Fig. 15C, 15D).
• AAV-mdys injected and AAV-mdys/Igf-I co-injected muscles displayed force generating capacities that were -47% and -49% of the values before the contractions, compared with -24% in mdx and -86% in wild-type animals (Fig. 15C).
• AAV-Igf-I treated animals retained -35% of the intial force generating capacity after the first lengthening confraction; however these values were not significantly different from mdx animals.
• AAV-Igf-I treated muscles were as susceptible to muscle damage as mdx control muscles and showed a force generating retention of only 3% (Fig. 15D).
• AAV-mdys treated, and AAV-mdys/Igf-I co-treated animals demonstrated a statistically significant 10% and 13% retention offeree generation, compared to 2% in mdx and 64% in wild-type animals.
• the values of AAV-mdys treated, and the AAV-mdys/Igf-I co-treated animals represent a 40% conection of the functional difference between mdx and wild-type muscles.
• AAV-mdys treated, and AAV-mdys/Igf-I co-treated animals revealed a 19% reduction in central nucleation relative to mdx, giving further evidence for the presence of fewer cycles of degeneration and regeneration in AAV-mdys treated TA muscles (Fig. 16A).
• central nucleation was analyzed selectively in dystrophin-expressing fibers, the percentage of centrally located nuclei decreased an additional 2-3% in comparison to random field analysis. Since reliable methods to directly visualize Igf-I expression in vivo are not available, analysis was limited to analyzing random fields, containing Igf-I transduced and non-transduced myofibers.
• AAV-Igf-I and AAV-mdys/Igf-I treatment both resulted in a -11% and -9% decrease in the total number of myofibers per unit area, which conelated with an increase in mean fiber diameter in AAV-Igf-I treated, and to a lesser extent in AAV-mdys/Igf-I co-treated, muscles compared with untreated mdx muscles.
• AAV-Igf-I and AAV-mdys/Igf-I demonstrated a higher absolute number of large muscle fibers than did AAV-mdys and mdx muscles (Fig. 16C).
• muscle fiber hypertrophy contributed to the increased muscle mass in AAV-Igf-I treated, and to a lesser extent in AAV-mdys/Igf-I, co-treated mdx muscles.
• AAV-mdys treated muscles demonstrated a small increase in mean fiber diameter that probably reflects a reduction in myofiber degeneration and thus the presence of fewer small caliber regenerating fibers (Fig. 16C).
• the total number of muscle fibers per unit area was compared to the total number of muscle fibers per muscle cross section.
• AAV-uDys treated animals demonstrated increased protection from contraction-induced injury, but did not display increases in mass or specific force.
• the combined treatment of both AAV-Igf-I and AAV-uDys showed an increase in muscle mass and strength together with protection from contraction-induced injury, indicating that co- treatment is more beneficial that treatment with either protein alone.
• EXAMPLE 8 Intravenous Administration of Igf-1 to Treat Symptoms of Aging
• This Example describes administration of Igf-1 to old mice in order to reduce muscle wasting type symptoms in old mice.
• Old mice (approximately 20 months old) were administered 1 x 10 12 vg/kg rAAV6-CK-Igf-l or rAAV6-CMV-Igf-l and were examined approximately 7 months after administration.
• Figure 17A shows intravenous administration of rAAV6-Igf-l vectors to old mice results in increased body mass consistent with increased muscle mass.
• Figure 17A shows the change in body mass for the control was approximately 3%, while the mice receiving Igf-1 was about 21% (CK promoter) and 18% (CMV promoter).
• Figure 17B show the change in Tibialis anterior (hindlimb) muscle mass for the control was about 48%, while mice receiving Igf-1 was about 52% (for both the CK and CMV promoters).
• Figure 18A shows intravenous administration of rAAV6-Igf-l vectors to old mice results in increased muscle strength and performance.
• Figure 18A shows the tibialis anterior (TA) muscles of treated old mice generate increased force compared with the muscles of untreated mice.
• Figure 18B shows that the muscles of treated mice display increased force output over a protocol of repetitive stimulation and at recovery. All publications and patents mentioned in the above specification are herein incorporated by reference.

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