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• • 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
• • 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
  • A61K48/0083—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 administration regime
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• ischemic heart disease is based on the bypass of diseased arterial segments with strategically placed bypass grafts (usually saphenous vein or internal mammary artery grafts).
• Percutaneous revascularization is based on the use of catheters to reduce the narrowing in diseased coronary arteries.
• FIGURE 1 is a schematic figure which shows rescue recombination construction of a transgene encoding adenovirus.
• %WTh percent wall thickening
• Another aspect of the present invention is a method for treating a heart disease in a patient having myocardial ischemia, comprising delivering a transgene-inserted vector to the myocardium of the patient by intracoronary injection, preferably by injecting the vector directly into one or both coronary arteries (or grafts), to transfect cardiac myocytes in the affected myocardium, said vector comprising a transgene coding for an angiogenic protein or peptide, for example, FGF-5, FGF-4, aFGF, bFGF or VEGF (vascular endothelial growth factor), and expressing the transgene in the heart, thereby promoting angiogenesis in the affected region of the myocardium.
• Other transgenes are also used to deliver a transgene-inserted vector to the myocardium of the patient by intracoronary injection, preferably by injecting the vector directly into one or both coronary arteries (or grafts), to transfect cardiac myocytes in the affected myocardium
• the present invention provides a method of production of a viral stock containing a recombinant vector capable of expressing an angiogenic protein or peptide in vivo in the myocardium, comprising the steps of cloning a transgene.
• a vasoactive agent for example histamine or a histamine agonist or a vascular endothelial growth factor (VEGF) protein
• telomeres preferably coding for an angiogenic protein or peptide such as FGF-4, FGF-5, aFGF, bFGF and VEGF.
• a polynucleotide containing a suitable signal sequence can be fused 5 ' to the first codon of the selected angiogenic protein gene.
• Suitable secretory signal sequences include signal sequences of the FGF-4, FGF-5, FGF-6 genes or a signal sequence of a different secreted protein such as IL-1 ⁇ .
• Angiogenic proteins can be modified to contain a signal sequence from another protein, for example by replacement of residues in the angiogenic protein with residues that direct secretion of the secreted second protein, as described in co-pending application U.S. Serial No. 08/852,779, filed May 6, 1997, incorporated by reference herein.
• a signal sequence derived from a protein that is normally secreted from cardiac myocytes can be used.
• genes encoding angiogenic proteins of human origin are preferred although angiogenic proteins of other mammalian origin, that exhibit cross-species activity i.e. having angiogenic activity in humans, can also be used.
• a gene of interest is transferred to the heart (or skeletal muscle), including cardiac myocytes (and skeletal myocytes), in vivo and directs constitutive production of the encoded protein.
• the helper-independent replication deficient human adenovirus system is a system that was used to transfer a gene of interest.
• myocardial cells in vivo by a single intracoronary injection.
• adenoviral vectors based on the human adenovirus 5 are missing essential early genes from the adenoviral genome (usually El A/El B), and are therefore unable to replicate unless grown in permissive cell lines that provide the missing gene products in trans.
• a transgene of interest can be cloned and expressed in tissue/cells infected with the replication deficient adenovirus.
• Retroviral vectors provide stable gene transfer, and high titers are now obtainable via retrovirus pseudotyping (Burns, et al., Proc Natl Acad Sci (USA) 90:8033-8037, 1993), but current retroviral vectors are unable to transduce nonreplicating cells (adult skeletal muscle and cardiac myocytes) efficiently.
• the potential hazards of transgene incorporation into host DNA are not warranted if short-term gene transfer is sufficient. Indeed, we have discovered that a limited duration expression of an angiogenic protein is sufficient for substantial angiogenesis. and transient gene transfer for cardiovascular disease and peripheral disease processes is therapeutically adequate.
• All adenoviral vectors used in the present invention can be constructed by the rescue recombination technique described in Graham. Virology 163:614-617, 1988.
• Plasmid JM17 encodes the entire human adenovirus 5 genome plus portions of the vector pBR322 including the gene for ampicillin resistance (4.3 kb). Although JM17 encodes all of the adenoviral proteins necessary to make mature viral particles, it is too large to be encapsulated (40 kb versus 36 kb for wild type).
• rescue recombination between the transgene containing the shuttle vector such as plasmid pACl and the plasmid having the entire adenoviral 5 genome such as plasmid pJM17 provides a recombinant genome that is deficient in the E1A/E1B sequences, and that contains the transgene of interest but secondarily loses the additional sequence such as the pBR322 sequences during recombination, thereby being small enough to be encapsulated (see Figure 1).
• we have reported successful results (Giordano, et al. Circulation 88:1-139, 1993, and Giordano and Hammond.
• adenovirus plasmids are also available from commercial sources, including, e.g., Microbix Biosystems of Toronto, Ontario (see, e.g., Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996). See also, the papers by Vile et al.. Nature Biotechnology, 15: 840-
• the present invention also contemplates the use of cell targeting not only by delivery of the transgene into the coronary artery, or femoral artery, for example, but also the use of tissue-specific promoters.
• tissue-specific transcriptional control sequences of left ventricular myosin light chain-2 (MLC :v ) or myosin heavy chain (MHC) to a transgene such as the FGF-5 gene within the adenoviral construct
• transgene expression is limited to ventricular cardiac myocytes.
• the efficacy of gene expression and degree of specificity provided by MLC 2V and MHC promoters with lacZ have been determined, using the recombinant adenoviral system of the present invention.
• the cardiac myocyte alone that is without concomitant expression in endothelial cells, smooth muscle cells, and fibroblasts within the heart
• an angiogenic protein such as FGF-5
• Successful recombinant vectors can be plaque purified according to standard methods.
• the resulting viral vectors are propagated on 293 cells which provide E1A and El B functions in trans to titers in the preferred 10 10 -10 12 viral particles/ml range.
• Cells can be infected at 80% confluence and harvested 48 hours later. After 3 freeze-thaw cycles the cellular debris is pelleted by centrifugation and the virus purified by CsCl gradient ultracentrifugation (double CsCl gradient ultracentrifugation is preferred).
• the viral stocks Prior to in vivo injection, the viral stocks are desalted by gel filtration through Sepharose columns such as G25 Sephadex.
• the final titer of such vector in the injectable preparation is preferably in the range of 10 7 -10 13 viral particles which allows for effective gene transfer.
• Other pharmaceutical carriers, formulations and dosages are described below.
• the vector transgene constructs are delivered to the myocardium by infusing one or both coronary arteries (or graft vessel) using standard percutaneous catheter based methods under fluoroscopic guidance, at an amount sufficient for the transgene to be expressed to a degree which allows for highly effective therapy.
• Gene delivery to other organs or tissues via intra-arterial infusion into an artery supplying the target tissue can be carried out in an analogous manner, preferably in combination with infusion of a vasoactive agent, for example histamine or a histamine agonist or a vascular endothelial growth factor (VEGF) protein, to enhance gene delivery as described and illustrated herein.
• a vasoactive agent for example histamine or a histamine agonist or a vascular endothelial growth factor (VEGF) protein
• vasoactive agent refers to a natural or synthetic substance that induces increased vascular permeability and enhances transfer of macromolecules such as gene delivery vectors across capillary walls.
• vasoactive agents can enhance delivery of these vectors to the targeted sites and thus effectively enhance overall expression of the transgene in the target tissue.
• histamine was employed as a vasoactive agent and was found to substantially enhance delivery of a vector to an infused site such as the myocardium.
• Histamine derivatives and agonists such as those that interact with the histamine H, receptor
• Histamine H, receptor include, for example, 2- methylhistamine, 2-pyridylethylamine, betahistine, and 2 thiazolylethylamine.
• These and additional histamine agonists are described, for example, in Garrison JC, Goodman and Gilman's The Pharmacological Basis of Therapeutics (8th Ed: Gilman AG, Rail TW, Nies AS, Taylor P, eds) Pergamon Press. 1990, pp 575-582.
• the strategy for therapeutic studies included the timing of transgene delivery, the route of administration of the transgene, and choice of the angiogenic gene.
• gene transfer was performed after stable but insufficient collateral vessels had developed.
• Previous studies using the ameroid model involved delivery of angiogenic peptides during the closure of the ameroid, prior to the development of ischemia and collateral vessels.
• this strategy was not employed for several reasons. First, previous studies are not suitable for closely duplicating the conditions that would be present in the treatment of clinical myocardial ischemia in which gene transfer would be given in the setting of ongoing myocardial ischemia; previous studies are analogous to providing the peptide in anticipation of ischemia, and are therefore less relevant.
• the vectors of the present invention such as the replication-deficient adenovirus allow for highly efficient gene transfer in vivo without cytopathic effect or inflammation in the areas of gene expression. Based on these results, described further in the below Examples, it is seen that a high enough degree of in vivo gene transfer to effect in vivo functional changes is achieved.
• the gene transfer of an angiogenic protein by intracoronary injection will promote angiogenesis and enhance cardiac function.
• treatment of cardiac ischemia can be conducted after observation of initial ischemic episodes.
• capillary number, blood flow and function will increase in the ischemic region.
• the entire dose of vector can be delivered via injection into the left coronary circulation (preferably LAD and LCx as noted above). Each injection is generally performed over a period of one to several minutes, typically about one and a half minutes.
• Histamine derivatives and agonists such as those that interact with the histamine H, receptor
• Histamine H, receptor include, for example, 2-methylhistamine, 2-pyridylethylamine, betahistine, and 2 thiazolylethylamine.
• These and additional histamine agonists are described, for example, in Garrison JC, Goodman and Gilman's The Pharmacological Basis of Therapeutics (8th Ed: Gilman AG, Rail TW, Nies AS, Taylor P, eds) Pergamon Press. 1990, pp 575-582.
• EXAMPLE 1 ADENOVIRAL CONSTRUCTS A helper independent replication deficient human adenovirus 5 system was used. The genes of interest were lacZ and FGF-5. The full length cDNA for human FGF-5 was released from plasmid pLTRI22E (Zhen. et al. Mol Cell Biol 8:3487.
• Recombinant adenoviruses encoding ⁇ -galactosidase, or FGF-5 were constructed using full length cDNAs.
• the system used to generate recombinant adenoviruses imposed a packing limit of 5 kb for transgene inserts.
• the genes proposed, driven by the CMV promoter and with the SV40 polyadenylation sequences were less than 4 kb. well within the packaging constraints.
• Recombinant vectors were plaque purified by standard procedures. The resulting viral vectors were propagated on 293 cells to titers in the 10 l0 -10 12 viral particles range. Cells were infected at 80% confluence and harvested at 36-48 hours.
• the cellular debris was pelleted by standard centrifugation and the virus further purified by double CsCl gradient ultracentrifugation (discontinuous 1.33/1.45 CsCl gradient: cesium prepared in 5 mM Tris. 1 mM EDTA (pH 7.8); 90,000 x g (2 hr). 105.000 x g (18 hr)).
• the viral stocks Prior to in vivo injection, the viral stocks were desalted by gel filtration through Sepharose columns such as G25 Sephadex. The resulting viral stock had a final viral titer in the 10"-10 12 viral particles range.
• the adenoviral construct was highly purified, with no wild-type (potentially replicative) virus.
• EXAMPLE 2 ADULT RAT CARDIOMYOCYTES IN CELL CULTURE
• tissue sections transmural sections of LAD bed 96 hours after intracoronary injection of adenovirus containing lacZ) revealed a significant magnitude of gene transfer observed in the LAD coronary bed with many tissue sections demonstrating greater than 50-60% of the cells staining positively for ⁇ -galactosidase.
• EXAMPLE 4 PORCINE ISCHEMIA MODEL Animals included 18 domestic pigs (30-40 kg). A left thoracotomy was performed under sterile conditions for instrumentation. (Hammond, et al. J Clin Invest 92:2644-2652, and Roth, et al. J Clin Invest 91 :939-949, 1993). Catheters were placed in the left atrium and aorta, providing a means to measure regional blood flow, and to monitor pressures. Wires were sutured on the left atrium to permit ECG recording and atrial pacing. Finally, an ameroid was placed around the proximal LCx.
• Studies were initiated 35 ⁇ 3 days after ameroid placement, at a time when collateral vessel development and pacing-induced dysfunction were stable (Roth, et al. Am JPhysiol 253 :H1279- 1288, 1987, and Roth, et al. Circulation 82:1778-1789).
• Conscious animals were suspended in a sling and pressures from the LV, LA and aorta, and electrocardiogram were recorded in digital format on-line (at rest and during atrial pacing at 200 bpm). Two dimensional and M-mode images were obtained using a Hewlett
• mice were anesthetized and midline thoracotomy performed.
• the brachycephalic artery was isolated, a cannula inserted, and other great vessels ligated.
• the animals received intravenous heparin ( 10,000 IU) and papaverine (60 mg).
• Potassium chloride was given to induce diastolic cardiac arrest, and the aorta cross-clamped.
• Saline was delivered through the brachycephalic artery cannula (120 mmHg pressure), thereby perfusing the coronary arteries.
• Glutaraldehyde solution (6.25%, 0.1 M cacodylate buffer) was perfused (120 mmHg pressure) until the heart was well fixed (10-15 min).
• the helper independent replication deficient human adenovirus 5 system constructed in Example 1 was used to prepare transgene containing vectors.
• the genes of interest were lacZ and FGF-5.
• the material injected in vivo was highly purified and contained no wild-type (replication competent) adenovirus. Thus adenoviral infection and inflammatory infiltration in the heart were minimized.
• Injection of the construct (4.0 ml containing about 10" viral particles of adenovirus) was made by injecting 2.0 ml into both the left and right coronary arteries (collateral flow to the LCx bed appeared to come from both vessels). Animals were anesthetized, and arterial access acquired via the right carotid by cut-down; a 5F Cordis sheath was placed. A 5F Multipurpose (A2) coronary catheter was used to engage the coronary arteries. Closure of the LCx ameroid was confirmed by contrast injection into the left main coronary artery. The catheter tip was then placed 1 cm within the arterial lumen so that minimal material would be lost to the proximal aorta during injection. This procedure was carried out for each of the pigs.
• a 5F Multipurpose (A2) coronary catheter was used to engage the coronary arteries. Closure of the LCx ameroid was confirmed by contrast injection into the left main coronary artery. The catheter tip was then placed 1 cm within the arterial lumen so that minimal material would
• FIGS. 4A-4C are diagrams corresponding to myocardial contrast echocardiographs.
• Figure 4A illustrates acute LCx occlusion in a normal pig, in which no flow is indicated in LCx bed (black) while septum (IVS) enhances (white), confirming that the image accurately identified the LCx bed and that reduced blood flow was associated with reduced contrast enhancement.
• Figure 4B illustrates the difference in contrast enhancement between IVS and LCx bed 14 days after gene transfer with lacZ, indicating different blood flows in two regions during atrial pacing (200 bpm).
• Figure 4C contrast enhancement appears equivalent in IVS and LCx bed 14 days after gene transfer with FGF- 5, indicating similar blood flows in the two regions during atrial pacing.
• Figure 3 summarizes computer analysis of video intensity in the two regions from all animals.
• data were expressed as the ratio of the peak video intensity (a correlate of myocardial blood flow) in the ischemic region (LCx bed) divided by the peak video intensity in the interventricular septum (IVS, a region receiving normal blood flow through the unoccluded left anterior descending coronary artery). Equal flows in the two regions would yield a ratio of 1.0.
• the ratio, prior to gene transfer, averaged 0.5, indicates substantially less flow in the LCx bed than in the septum.
• Figure 3 shows that animals receiving lacZ gene transfer had a persistent blood flow deficit in the ischemic region.
• Electropherograms upon the PCR amplification confirmed the presence of JM17- CMV-FGF-5 DNA (the expected 500 bp fragment) in the LAD and LCx beds of three pigs 14 days after gene transfer with FGF-5. Electropherogram upon the RT-PCR amplification confirmed the presence of cardiac FGF-5 mRNA (the expected 400 bp fragment) in the
• EXAMPLE 5 USE OF A VASOACTIVE AGENT TO ENHANCE GENE DELIVERY
• a vector such as a viral vector
• a vasoactive agent such as histamine
• histamine as an exemplary vasoactive agent
• histamine infusion of the coronary artery dramatically increased the gene transfer efficiency. Briefly, a pig was anesthetized and arterial access obtained via the right carotid artery. A 5 French multipurpose catheter was placed in the right coronary artery (RCA) and inserted 2 cm within the ostia.
• the catheter was then removed from the RCA and placed in the left anterior descending coronary artery (LAD), again inserting the catheter tip 2 cm within the LAD lumen.
• LAD left anterior descending coronary artery
• histamine infusion was performed within several minutes prior to the delivery of vector.

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