JP2002533359A - Efficient and stable in vivo gene transfer into cardiomyocytes using recombinant adeno-associated virus vectors - Google Patents

Abstract

  (57) [Summary] The present invention relates to the use of a recombinant adeno-associated virus (rAAV) vector to transduce cardiomyocytes in vivo by injecting rAAV into the coronary artery or coronary sinus. rAAV infection is not accompanied by detectable myocardial inflammation or muscle cell necrosis. Thus, rAAV is a useful vector for the stable expression of therapeutic genes in the myocardium, and introduces angiogenesis, inhibits angiogenesis, stimulates cell proliferation, inhibits cell proliferation and / or other cardiovascular conditions Can be used to deliver genes for the treatment or improvement of

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is a continuation-in-part of U.S. Provisional Patent Application No. 60 / 113,923, filed December 28, 1998, which is incorporated herein by reference in its entirety. Is done.

[0002] This study is based on the National Institutes of Health (
DK-48997, AR-42895, and HL-54592) to Jeff
rey M. Partially supported by a grant to Leiden.

[0003] The ability to stably and efficiently program recombinant gene expression in cardiomyocytes facilitates gene therapy approaches for various cardiovascular diseases and conditions. Thus, the present invention relates to the use of rAVV vectors to transduce cardiomyocytes in vivo by injecting a recombinant adeno-associated virus (rAAV) vector into the coronary artery or coronary sinus. For example, coronary perfusion of mouse heart with rAAV encoding the LacZ gene resulted in efficient transduction of cardiomyocytes, which was stable for at least 8 weeks. Furthermore, rAAV infection is not accompanied by detectable myocardial inflammation or muscle cell necrosis. Thus, rAAV is a useful vector for stable expression of therapeutic genes in the myocardium and induces angiogenesis, inhibits angiogenesis, stimulates cell proliferation, inhibits cell proliferation and / or other cardiovascular It can be used to deliver genes for treatment or amelioration of a condition.

BACKGROUND OF THE INVENTION [0004] Gene therapy of the myocardium can be used for the treatment of a number of cardiovascular diseases, including ischemic cardiomyopathy, congestive heart failure, and malignant arrhythmias (Nabel (1995)).
Circulation 91: 541-548). Useful vectors for myocardial gene delivery enable efficient and stable transduction of cardiomyocytes with various transgenes, either after direct intramyocardial injection or injection into the coronary artery or coronary sinus To For example, a plasmid DNA vector injected directly into the left ventricular myocardium was expressed by cardiomyocytes near the area of injection for more than six months (Lin et al. (19).
90a) Circulation 82: 2217-2221; Kass et al. (1
993) Proc. Natl. Acad. Sci. USA 90: 11498-
11502; and Guzman et al. (1993) Circ. Res. 73:12
02-1207). However, the therapeutic utility of this approach has been limited by the low efficiency of cardiomyocyte transduction (0.1% to 1.0% of cardiomyocytes in the area of injection).

[0005] Intramyocardial and intracoronary injection of replication-defective adenovirus (RDAd) vectors have been used in rodents, rabbits, and pigs to efficiently transduce cardiomyocytes in vivo. However, the potential for adenovirus-mediated gene transfer is limited by the immune response to the viral and foreign transgene proteins, which causes significant myocardial inflammation and eliminates viral transduced cells within 30 days of infection. And thereby results in transient recombinant gene expression in immunocompetent hosts (Guzman et al. (1993) Ci.
rc. Res. 73: 1202-1207; French et al. (1994) Cir.
circulation 90: 2414-2424; and Barr et al. (1994).
Gene Ther. 1: 51-58).

[0006] Recently, rAAV vectors have been used in both rodents and primates in skeletal muscle and liver (Fisher et al. (1997) Nat. Med. 3: 306-312; Kes.
Sler et al. (1996) Proc. Natl. Acad. Sci. USA 93
: 14082-14087; and Snyder et al. (1997) Nat. Gen
et. 16: 270-276) and myocardium directly injected with rAAV (Pod).
Sakoff et al., US Pat. No. 5,858,351) have been shown to program efficient and stable recombinant gene expression. However, rAAV vectors used in gene therapy applications, unlike RDAds, do not encode a viral protein, and thus do not involve an immune response to the foreign transgene protein.

[0007] Previous reports have shown that rAAV can transduce cardiomyocytes in vivo, but the efficiency of rAAV-mediated transgene expression in the heart is low (about 0.2%).
And localization (Kaplitt et al. (1996) Ann. Thorac. Surg.
62: 1669-1676). In this study, porcine hearts were rapidly perfused with low titers of rAAV (less than 10 ⁴ expression units AAV / g body weight). Based on these results, infusion of rAVV into the heart severely limits its use as a vector for myocardial gene therapy. However, as shown herein, the present invention provides a method for injecting rAAV at very high doses and at specific times proportional to the body weight of the animal, which then leads to an unexpectedly efficient and stable cardiac To establish the use of rAAV vectors to deliver therapeutically effective molecules to the myocardium in amounts effective to treat or ameliorate a cardiac disease or condition.

SUMMARY OF THE INVENTION [0008] The present invention provides rAAV in a coronary artery or coronary sinus in a time and amount sufficient to stably and efficiently transduce cardiomyocytes perfused by the coronary artery or coronary sinus.
A method for treating a cardiovascular condition by injecting a vector. This rA
The AV vector encodes at least one nucleic acid operably linked to a control region and encoding a therapeutically effective molecule. After injection and cardiomyocyte transduction, the therapeutically effective molecule is expressed in an amount effective to treat or ameliorate the cardiovascular condition.

[0009] Thus, the method provides a means for delivering AAV vectors in a stable and efficient manner. The vector can be injected by any convenient means, and if desired, with surgery or other cardiac procedures.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the treatment of cardiovascular conditions using rAAV vectors. In accordance with the present invention, a rAAV vector encoding a therapeutically effective molecule is injected into the coronary artery or coronary sinus and delivers the vector to the heart in a manner that stably and effectively transforms cardiomyocytes. Unexpectedly, the ability to obtain stable and effective transformation of cardiomyocytes with rAAV was found to be dependent on the length of the infusion period and the amount of virus injected relative to body weight.

[0011] In addition, rAAV shows significant advantages for myocardial gene transfer as compared to plasmid DNA or adenovirus vectors. For example, rAAV, when delivered as described herein, allows for efficient transformation of cardiomyocytes. In addition, rAAV vectors program stable expression of foreign transgenes in immunocompetent hosts. The stability of transgene expression observed with rAAV even after expression of the foreign transgene protein is that rAAV vectors, unlike their adenovirus counterparts, do not express any viral gene products and are therefore significantly immunogenic Seems to reflect the fact that This lack of immunogenicity indicates a major advantage of rAAV for myocardial gene transfer.

Thus, the present invention is directed to a method of treating a cardiovascular condition. The method involves injecting an rAAV vector into a coronary artery or sinus of an animal for a time and in an amount sufficient to stably and efficiently transform cardiomyocytes perfused by the artery or sinus. Here, the vector comprises a transgene encoding at least one nucleic acid (ie, a transgene encoding a therapeutically effective molecule; and a transgene expressing the therapeutically effective molecule in an amount effective to treat or ameliorate a cardiovascular condition. Gene).
Further, the nucleic acid is operably linked to control regions that allow expression of the molecule (eg, promoters, enhancers, termination signals, etc.). If more than one nucleic acid is present on the rAAV vector, each may be separately controlled by an individual control region, or any group of them or all of them may be in one operon (ie, a single transcript). (A single control region that drives the expression of multiple genes).

[0013] rAAV vectors useful in the present invention include appropriate regulatory elements (eg,
Any rAAV vector having one or more transgenes (or nucleic acids of interest) inserted into the vector in a manner that allows for expression of the transgene under the control of a promoter, enhancer, transcription terminator, etc. obtain. rAAV vectors are well known in the art and can be prepared by standard methodology known to those skilled in the art. For example, US Pat. No. 5,858,351 and references cited therein describe various rAAV vectors suitable for use in gene therapy, as well as methods for making and propagating those vectors (eg, Kotin).
(1994) Human Gene Therapy 5: 793-801 or Berns "Parvoviridae and theair Repli.
station "Fundamental Virology, 2nd edition (Field
ds & Knipe, eds.)).

“Transgene” or “nucleic acid of interest” or “nucleic acid encoded in an rAAV vector” as used herein, is a therapeutically effective molecule that can be used to treat a cardiovascular condition. Refers to any nucleotide sequence that encodes Such transgenes will usually be foreign to the animal to be treated, or it is desired that the altered expression (eg, expression time, expression space, or expression amount) achieves a particular therapeutic effect. May be a gene normally found in animals. The therapeutically effective molecule encoded by the transgene is a protein, or antisense RNA, that confers an advantage to an animal or subject undergoing treatment or amelioration of a cardiac condition or disease, according to the present invention.

[0015] There are many proteins that can be administered to treat or ameliorate a cardiovascular condition, and include, but are not limited to: molecules suitable for inducing angiogenesis (eg, angiogenic factors) Anti-angiogenic factors; proteins capable of inhibiting vascular smooth muscle cell proliferation; proteins useful for treating atherosclerosis; proteins useful for treating restenosis; for stimulating cardiomyocyte activity Useful proteins; proteins secretable from cardiomyocytes (which exert their effects in the heart) or proteins that can be transported elsewhere for the treatment of cardiovascular conditions or diseases; useful in the treatment of heart conditions or diseases Hormones, cytokines or growth factors; and proteins that can stimulate vascular smooth muscle cell proliferation. Other genes encoding proteins useful in the present invention include ion channel genes,
Contractile protein genes, phospholamban-encoding genes and genes encoding β-adrenergic receptors or β-adrenergic kinases.

Examples of blood vessel-derived factors include FGF-1, FGF-2, FGF-5, VEGF,
HIF-1 and the like, but are not limited thereto. Proteins useful for treating restenosis include thymidine kinase, cytosine deaminase, p21, p2
7, p53, Rb and NF-κB. Thus, the present invention can be used to deliver any protein via an rAAV vector that has therapeutic benefits to treat or ameliorate a cardiovascular condition or disease.

[0017] Proteins suitable for inducing angiogenesis, ie, “angiogenic factors”
As used herein, a protein or substance that causes the growth of new blood vessels, and includes fibroblast growth factor, endothelial cell growth factor or other proteins having such biological activity. Particular proteins known to induce angiogenesis are FGF-1, FGF-2, FGF-5, VEGF and their active fragments, and HIF-1. Proteins suitable for inhibiting angiogenesis, ie, “anti-angiogenic factors”, are proteins or substances that inhibit the formation of new blood vessels.

[0018] Antisense RNA that can be administered to treat or ameliorate a cardiovascular condition
Has one of the same activities as proteins useful in the present invention. Such R
Examples of NA include c-myb and c-myc, but are not limited thereto. Antisense RNA molecules, including methods for designing and using such molecules in expression vectors, are well known in the art and can be constructed by conventional methodology. Thus, an RNA strand whose base sequence is complementary to a sense (or translated) RNA strand blocks translation or degradation of a particular mRNA (otherwise controls or alters expression of a desired mRNA). To form a double strand.

As used herein, “regulatory region” or “regulatory element” refers to a polyadenylation signal that regulates transcription and translation of a nucleic acid sequence, upstream regulatory domains, promoters, enhancers, transcription termination sequences, and the like.

The term “operably linked” refers to a sequence of elements. Here, the components are arranged to perform their normal function. Thus, a control region or regulatory element operably linked to a coding sequence can affect the expression of the coding sequence. A control element need not be adjacent to a coding sequence as long as it functions to direct its expression. Thus, for example, the intervention of an untranslated but transcribed sequence may be between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence. Can be done.

[0021] The regulatory elements of the present invention can be from any source, such as viruses, mammals, insects or even synthetic, provided that they function in cardiomyocytes. For example, any promoter can be used to control the expression of the transgene. Such promoters can be promiscuous (ie, active in many cell types) (eg, SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP),
Herpes simplex promoter, CMV promoter (eg, CMV immediate early promoter, Rous sarcoma virus (RSV) promoter). Alternatively, the promoter may be tissue specific for expression in cardiomyocytes.

This rAAV is delivered to cardiomyocytes by injection into the coronary artery or coronary sinus. This mode of delivery has also been referred to as intravascular, intracoronary or intraarterial delivery through the coronary arteries. As used herein, infusion into the coronary artery includes intracoronary perfusion. In accordance with the present invention, the rAAV vector is used when the heart is in situ (
(I.e., within a body cavity) or if the heart or heart tissue has been removed from the body (as occurs when a heart is provided for transplantation into a recipient). In the case of a heart that has been prepared for transplantation or for cardiac tissue, the vector may be contacted with or immersed in a suitable concentration of a solution of the vector, or With a combination of both, it can be infused through any artery or vein in contact with the heart. Thus, as described herein, infusion includes delivering the rAAV to the heart or heart tissue ex vivo by the means disclosed herein. If necessary, the infusion can be repeated, for example, at three months, six months, one year, or at intervals as determined appropriately.

As used herein, treating a cardiac condition includes treating a cardiac or cardiovascular disease. Examples of cardiac conditions that may be treated or ameliorated in accordance with the methods of the invention include myocardial ischemia, myocardial infarction, congestive heart failure, dilated and hypertrophic cardiomyopathy, cardiac arrhythmia, cardiac hypertrophy, cardiac hypertrophy Including but not limited to transplantation and cardiac rejection. For example,
If a cardiac condition such as ischemia can be treated or ameliorated by inducing angiogenesis, then the rAAV vector used in accordance with the methods of the invention encodes an angiogenic factor.

Thus, the rAAV vector is injected into the coronary arteries in a temporary and sufficient amount to stably and effectively transduce cardiac tissue perfused by the artery, where the AAV vector is Treat or ameliorate the condition (induce angiogenesis, inhibit angiogenesis, stimulate or inhibit cell proliferation, treat restenosis, treat atherosclerosis, treat congestive heart failure, treat ischemic cardiomyopathy or malignant Arrhythmia (ma
therapeutically effective molecule expressed in heart tissue in an amount effective to (including, but not limited to, treatment of myocardial infarction, congestive heart failure, or dilated and hypertrophic cardiomyopathy). Code.

The method of the present invention can be used with any animal, including but not limited to mammals such as rodents, dogs, cats, cows, primates, and humans. Preferably, the method is used for gene therapy to treat a human acquired or congenital heart condition or disease.

Accordingly, the present invention provides a method for treating and / or ameliorating a cardiovascular condition by injecting a rAAV vector in a transient and sufficient amount to stably and effectively transduce cardiomyocytes. Provided, which have heretofore been unable to be achieved by methods known in the art. For the purposes of the present invention, stable and effective transduction means that a significant number of cardiomyocytes have been transduced and are capable of expressing the protein for an extended period of time. Stable and effective transduction occurs over a long period of time, and as sustained expression of the transgene, as an increase in the percentage of transduced cardiomyocytes,
Or, it can actually be observed over time, as a sustained observation of the therapeutic effect at the molecular, microscopic, or macroscopic level. For example, stable and effective transduction with angiogenesis is due to the ongoing development and or proliferation of new blood vessels.
It can be revealed by observing the improved vascular flow to the heart or by determining the measured level of ischemia in heart tissue.

Alternatively, efficient transduction occurs when at least about 10% and preferably more cardiomyocytes are transduced (ie, infected by rAAV). By following the methods of the invention and by observing at specific times after transduction for several weeks to many weeks, up to about 25%, about 40% or even about 50% of the cardiomyocytes are transduced. Approximately 10% of cardiomyocytes can be transduced using rAAV alone, but this percentage can be increased by co-injecting adenovirus as a helper virus without adverse effects.

[0028] The time of injection also contributes to achieving stable and effective transduction of cardiomyocytes. Thus, the infusion time ranges from about 2 minutes to about 30 minutes, more preferably from about 5 minutes to about 20 minutes, and most preferably about 15 minutes.

The amount of rAAV injected into an animal is proportional to the animal's body weight. Thus, consistent with the present invention, stable and effective transduction indicates that the amount of rAAV injected is
About 1 × 10 ⁵ IU per gram (infectious unit)
) To about 1 x 10 ⁹ IU of AAV per gram of body weight, and preferably from about 1 x 10 ⁶ IU per gram of body weight to about 1 x 10 ⁸ IU of AAV per gram of body weight, and most preferably, 1 About 5-6 × 10 ⁷ IU AA per gram
V.

The example described below demonstrates the efficient and stable transduction of cardiomyocytes in vivo after coronary injection of rAAV vector.

Throughout this application, various publications, patents, and patent applications are referenced. The teachings and disclosures of these publications, patents, and patent applications, in their entireties, are hereby incorporated by reference into this application to better describe the state of the art to which this invention pertains. .

It is understood and expected that modifications in the principles of the invention herein disclosed in the exemplary embodiments may be made by those skilled in the art, and that such modifications, changes, and substitutions It is intended to be included within the range.

(Example) (Intracoronary injection of rAAV) (1. Method :) (Plasmid and virus) The structure of pAAV _CMV-LacZ is shown in FIG. The Ad _CMV-LacZ and E3-deleted adenovirus, Ad _d1309, was _prepared as described in (Barr 1994)
Propagated and purified.

(Amplification and Purification of rAAV) rAAV was purified from (Rolling et al. (1995) Mol. Biotechnol).
. 3: 9-15) and purified by cesium chloride gradient centrifugation. Viral titer was determined by dot blot hybridization assay to determine the number of viral genomes per milliliter, and the virus was used to infect HeLa cells and X-gal 24 hours after infection. It was evaluated by staining. All virus preparations contained 1-2 × 10 ¹¹ genomes / mL, and 2-3 × 10 ⁹ infectious units (I
U) / mL.

(Intracoronary Perfusion with rAAV) Adult C57BL / 6 mouse hearts were treated with a cardioplegic solution (110
mmol / L NaCl, 25 mmol / L KCl, 22 mmol / L Na
HCO ₃ , 16 mmol / L MgCl ₂ , 0.8 mmol / L CaCl ₂ , 4
(0 mmol / L glucose) at 4 ° C. until the heart stopped beating. They were then brought to ex vivo for 15 minutes at a rate of 33 μL / min.
. Perfused with 1.5 × 10 ⁹ IU of AAV _CMV-LacZ in 5 mL of PBS at 4 ° C. After perfusion, the heart is implanted in the neck of the syngeneic host, the donor artery is anastomosed to the host's right common carotid artery, and the donor's pulmonary artery is connected to the right external jugular vein.
Ugular vein (Lin et al. (1990b) J. Heart).
Transplant. 9: 720-723) (n = 3 for each time point)
.

(X-gal staining) Freshly isolated hearts were fixed in PBS supplemented with 1.25% glutaraldehyde for 10 minutes at room temperature and stained with X-gal (Lin 1990a) overnight. And counterstained with eosin.

(Β-Galactosidase Activity) Heart homogenates were evaluated for β-galactosidase (β-gal) activity and protein concentration. β-Gal activity was normalized for total protein and for the number of infectious rAAV or RDAd particles injected.

II. Results Many clinical applications of myocardial gene therapy may require stable and efficient transduction of cardiomyocytes distributed over large areas of the myocardium. Injection of coronary arteries with RDAd has been shown to result in effective transduction of cardiomyocytes throughout the area of perfused myocardium (Barr 1994). To test whether rAAV could similarly transduce cardiomyocytes after perfusion of the coronary arteries, transplant hearts from C57BL / 6 mice and place them via a catheter placed in the left common carotid artery. For 15 minutes at 4 ° C
_Was perfused with 1.5 × 10 ⁹ IU of AAV _CMV-LacZ . These perfused hearts were then transplanted into syngeneic hosts and arterial circulation was reestablished by anastomosis of the aorta implanted in the recipient's carotid artery. Such transplanted and revascularized hearts resume beating and 2 perfusion of recipient mice.
The beating continued until killing in 4, or 8 weeks. Two weeks after perfusion, a small number (<1
%) Β-gal-positive cardiomyocytes were detected throughout the myocardium of rAAV-perfused hearts (FIG. 2). By 4 weeks after perfusion, about 40% of cardiomyocytes were β-gal positive. This high level of transduction stabilized after a few weeks after perfusion, with> 50% of cardiomyocytes continuing to express β-gal. Similar increases in expression of recombinant genes over the first few weeks after rAAV infection were observed in skeletal muscle (Fisher 199).
7; Kessler 1998). Such an increase is due to the transgene (trans
It has been speculated that the transcriptional ability of C.gene. may affect the gradual process of conversion of a single-stranded AAV genome into a double-stranded DNA molecule (Ferrari et al. (19).
96) J. Amer. Virol. 70: 3227-3234). Thus, rAAV delivered by coronary artery perfusion can be used to stably transduce cardiomyocytes throughout the myocardium.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of AAV _CMV-LacZ . ITR stands for inverted terminal repeat; BGH
pA indicates bovine growth hormone polyadenylation signal; CMV Pr indicates CMV immediate early promoter; LacZ indicates bacterial LacZ gene.

FIG. 2 shows in vivo gene transfer into cardiomyocytes using AAV _CMV-LacZ . Coronary artery perfusion with 1.5 × 10 ⁹ IU AAV _CMV-LacZ and X-gal
FIG. 4 shows a total cross section (left) and a micrograph (right) of a mouse heart after staining with the mouse. The length of the bar is 25 microns.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 9/10 A61P 9/10 101 101 C12N 15/09 A61K 35/76 // A61K 35/76 C12N 15 / 00 A 38/00 A61K 37/43 38/04 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K) E, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, U, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Svenson, Eric United States Illinois 60637, Chicago, South Maryland 5841, Department of Medicine, University of Chicago F-term (reference) 4B024 AA01 AA20 DA03 EA04 FA20 HA17 4C084 AA02 AA13 BA44 DB54 DB55 DB62 MA 01 NA05 NA14 ZA362 ZA372 ZA392 ZA402 ZA452 ZB212 4C086 AA01 AA02 EA16 MA01 MA04 NA05 NA14 ZA36 ZA37 ZA39 ZA40 ZA45 ZB21 4C087 AA01 AA02 AA03 BB65 BC83 MA01 NA05 NA14 ZA36 ZA37 ZA39 ZA37

Claims (17)

[Claims] 1. A method for producing a medicament for treating a cardiovascular condition by expressing a therapeutically effective molecule in an amount effective to treat or ameliorate the cardiovascular condition,
Use of a recombinant adeno-associated virus (AAV) vector encoding at least one nucleic acid operably linked to a control region, said nucleic acid encoding said therapeutically effective molecule, wherein said medicament is The use, wherein the cardiovascular artery or sinus is infused into the coronary artery or sinus for a time and amount sufficient to stably and efficiently transduce cardiomyocytes perfused by the coronary artery or sinus. 2. The use of claim 1, wherein said AAV transduces at least about 10% of said cardiomyocytes. 3. The use of claim 2, wherein said AAV transduces at least about 40% of said cardiomyocytes. 4. The use of claim 3, wherein said AAV transduces at least about 50% of said cardiomyocytes. 5. The use of any one of claims 1-4, wherein the AAV is infused for at least about 2 minutes to about 30 minutes. 6. The use of claim 5, wherein the AAV is infused for at least about 5 minutes to about 20 minutes. 7. The use of claim 6, wherein the AAV is infused for about 15 minutes. 8. The amount of the AAV is about 1 × 10 ⁵ IU A / g body weight.
AV~1g is weight per about 1 × 10 ⁹ IU AAV, Use according to any one of claims 1 to 7. 9. The amount of AAV is about 1 × 10 ⁶ IU A / g body weight.
AV~1g is weight per about 1 × 10 ⁸ IU AAV, Use according to claim 8. 10. The amount of AAV is about 6 × 10 ⁷ IU / g body weight.
The use according to claim 9, which is AAV. 11. The coronary artery is injected ex vivo or in vivo,
Use according to any one of claims 1 to 10. 12. The use according to any one of claims 1 to 11, wherein said therapeutically effective molecule is an antisense RNA or protein. 13. The therapeutically effective molecule is an ion channel gene, a contractile protein, a phospholamban, a β-adrenergic receptor, a β-adrenergic kinase, a growth factor, an angiogenic factor, a protein or nucleic acid capable of inducing angiogenesis, or an angiogenesis The use according to any one of claims 1 to 11, which is a protein or a nucleic acid capable of inhibiting the following. 14. The therapeutically effective molecule is FGF-1, FGF-2, FGF.
The use according to any one of claims 1 to 11, which is -5, VEGF or HIF-1. 15. The therapeutically effective molecule may be thymidine kinase, p21, p2.
Use according to any one of claims 1 to 11, which is 7, p53, Rb or NF-κB. 16. The cardiovascular condition is restenosis, atherosclerosis, congestive heart failure, ischemic cardiomyopathy, malignant arrhythmia, myocardial infarction, or dilated and hypertrophic cardiomyopathy. Use according to any one of claims 1 to 11. 17. The method for treating or ameliorating a cardiovascular condition comprises inducing angiogenesis, inhibiting angiogenesis, stimulating or inhibiting cell proliferation, treating restenosis, treating atherosclerosis, treating congestive heart failure. The use according to any one of claims 1 to 11, which is a treatment or improvement by treatment of ischemic cardiomyopathy or treatment of malignant arrhythmia.