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  • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
  • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

• the present invention relates to a recombinant adenovirus vector which is used in gene therapy for myocardial ischemia, a method for producing same, and a method of providing myocardial protection during revascularization or non-revascularization procedures with the use of the vector.
• the vector efficiently expresses a transgene in the myocardium.
• Myocardial ischemia occurs when the heart muscle does not receive an adequate blood supply and is thus deprived of necessary levels of oxygen and nutrients.
• the most common cause of myocardial ischemia is atherosclerosis, causing blockages in the blood vessels (coronary arteries) that provide blood flow to the heart muscle.
• Present treatment modalities include pharmacologic therapies, coronary artery bypass surgery and percutaneous revascularization using techniques such as balloon angioplasty.
• Decreased demand of the heart muscle for oxygen and nutrients is accomplished either by agents that decrease the hemodynamic load on the heart or those that decrease the contractile response of the heart to a given hemodynamic load.
• Surgical treatment of ischemic heart disease is based on the bypass of diseased arterial segments with strategically placed bypass graft (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. All of these strategies are based on the eradication of ischemic episodes as the primary treatment evidence, and all have limitations of their effectiveness in this regard.
• Australian Patent Publication No. 27902/92 discloses adenovirus vectors for expression of desired genes in muscle cells to treat muscular dystrophy and thromboses.
• the '902 application discloses adenovirus type 5
• it discloses specific vector constructs which are used for the treatment of muscular dystrophy.
• Texas Heart Institute Journal article 21:104-11 (1994) discloses the advantages of use of adenoviral vectors in mediating efficient direct gene transfer for preventing restenosis.
• this article teaches that the Ad5 virus transfected into 293 cells is an extremely useful vector for gene transfer in coronary arteries.
• none of the treatment modalities of the prior art addresses the issue of protection of the myocardium against irreversible damage when ischemia does occur.
• Protection of heart muscle against ischemia has been demonstrated in the setting of ischemic pre-conditioning. This phenomenon occurs when the heart is exposed to brief periods of ischemic stress prior to a prolonged ischemic episode. During the brief periods of ischemia, production of specific stress related factors is induced. These stress factors protect the myocardium against subsequent and potentially more harmful, prolonged ischemic episodes. To date, attempts to induce these same factors by pharmacologic means have been unsuccessful.
• FIGURE 1 is a schematic figure which shows rescue recombination construction of a transgene encoding adenovirus.
• FIGURE 2 schematically presents the strategy for introducing a foreign gene into the El region of a replication-deficient adenoviral vector.
• FIGURE 3 graphically presents the lactate dehydrogenase released by adenoviral infected H9c2 cells following simulated ischemia.
• FIGURE 4 graphically presents the creatine kinase released by adenoviral infected neonatal rat myocytes following simulated ischemia.
• the present invention has exploited a gene therapy approach to treat heart disease.
• An objective of the present invention is to provide a method of providing myocardial protection in which a stress related factor is produced in the myocardium and is present at the time of ischemia so as to protect the myocardium against subsequent, potentially more harmful, prolonged ischemic episodes. This objective concerns protective effects, rather than therapeutic effects on myocardial ischemia.
• one important aspect of the present invention is a method of providing myocardial protection, comprising: delivering a replication-deficient adenoviral vector to a myocardium by intracoronary injection into the coronary arteries, preferably a single injection of the vector, directly into the coronary arteries, so as to transfect cardiac myocytes, which do not undergo rapid turnover, in the affected myocardium, said vector comprising a transgene coding for a stress related factor such as heat shock proteins HSP70i, HSP27, HSP40 and HSP60, and the adenosine A3 receptor; and expressing the transgene in the myocardium, thereby raising the level of stress related factor in the affected region of the myocardium.
• a stress related factor such as heat shock proteins HSP70i, HSP27, HSP40 and HSP60
• the myocardium By injecting the vector stock containing no wild-type virus deeply into the lumen of the coronary arteries, preferably into both the right and left coronary arteries, of the myocardium preferably in an ischemic milieu, preferably in an amount of 10 10 -10 13 viral particles as determined by optical densitometry (more preferably 10 n -10 12 viral particles), it is possible to locally transfect most of the cells, especially cardiac myocytes, which do not undergo rapid turnover, in the affected myocardium with the genes for a stress related factor, thereby maximizing myocardial protection efficacy of gene transfer, and minimizing the possibility of an inflammatory response to viral proteins. If a ventricular myocyte-specific promoter is used, the promoter more securely enables expression limited to the cardiac myocytes so as to effectively avoid the potentially harmful effects of angiogenesis in non-cardiac tissues such as the retina.
• myocardial protection is expected to be more effective in cases that (a) said patient has non-revascularized ischemic heart disease and said protection is desired during planned non-cardiac surgery, wherein said vector is administered a plurality of days prior to the planned non- cardiac surgery; (b) said protection is desired in anticipation of complex percutaneous revascularization, and wherein said vector is delivered at the time of a diagnostic catheterization a plurality of days prior to the revascularization; (c) said protection is desired in anticipation of complex cardiac surgery, and wherein said vector is delivered at the time of a diagnostic cardiac catheterization; (d) said protection is desired in a donor heart to be transplanted into a host patient with a coronary disease, and wherein said vector is delivered at the time of a diagnostic coronary angiography prior to explanation to rule out coronary disease; and (e) said protection is desired in a patient with diffuse, nonrevascularizable coronary artery disease, at the time of a diagnostic coronary angiography prior to explanation to rule out
• an injectable adenoviral vector preparation comprising a recombinant adenoviral vector, preferably in a final viral titer of 10 10 -10 12 viral particles, said vector containing no wild-type virus and comprising a partial adenoviral sequence from which the E1A/E1B genes have been deleted, and a transgene coding for a stress related factor such as heat shock proteins HSP70i, HSP27, HSP40 and HSP60, and the adenosine A3 receptor, driven by a promoter flanked by the partial adenoviral sequence; and a pharmaceutically acceptable carrier.
• a stress related factor such as heat shock proteins HSP70i, HSP27, HSP40 and HSP60
• adenosine A3 receptor driven by a promoter flanked by the partial adenoviral sequence
• a pharmaceutically acceptable carrier By using this injectable adenoviral vector preparation, it is possible to perform effective adenovirus- mediated stress related factor-coding
• a further aspect of the present invention is a method of production of a viral stock containing a recombinant vector capable of expressing a stress related factor in vivo in the myocardium, comprising the steps of cloning a transgene coding for a stress related factor such as heat shock proteins HSP70i, HSP27, HSP40 and HSP60, and the adenosine A3 receptor into a plasmid containing a promoter and a polylinker flanked by partial adenoviral sequences of the left end of the human adenovirus 5 genome from which the E1A/E1B genes have been deleted; co-transfecting said plasmid into mammalian cells transformed with the E1A/E1B genes, with a plasmid which contains the entire human adenoviral 5 genome and an additional insert making the plasmid too large to be encapsulated, whereby rescue recombination takes place between the transgene-inserted plasmid and the plasm
• various stress related factors which are capable of protecting myocardial ischemia can be used; heat shock proteins HSP70i, HSP27, HSP40 and HSP60, and the adenosine A3 receptor can be exemplified.
• Adenosine plays an important role in mediating the phenomenon of ischemic preconditioning. The function of adenosine appears to be mediated via A3 type adenosine receptors. In cell culture experiments in which the number of A3 receptors per cell was increased, the efficacy of an adenosine analogue (Gensia Pharmaceutical) to mitigate protection against ischemia was increased.
• coding regions for these factors are known in the art, and it is possible to download these cDNA sequences from Genebank and other databanks over the internet, for example. Full or partial length cDNAs coding for the above factors can be used in the present invention. Other than above, sarcoplasmic reticular calcium ATPase can be used for the purpose of studying myocardial calcium handling/hypertrophy.
• helper Independent Replication Deficient Human Adenovirus 5 System The cDNA of interest is transferred to the myocardium, including cardiac myocytes, in vivo and directs constitutive production of the re-encoded protein.
• Viral vectors provide a means for highly efficient gene transfer. Several different gene transfer approaches are feasible. The present inventors initially used the helper-independent replication deficient human adenovirus 5 system which has previously demonstrated transfection greater than 60% of myocardial cells in vivo by a single intracoronary injection. Non-replicative recombinant adenoviral vectors are particularly useful in transfecting coronary endothelium and cardiac myocytes resulting in highly efficient transfection after intravenous injection.
• adenoviral vectors based on the human adenovirus 5 ⁇ Virology, 163:614-617 (1988) ⁇ are missing essential early genes from the adenoviral genome (usually E1A/E1B), 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 will be expressed in tissue/cells infected with the replication deficient adenovirus.
• adenovirus-based gene transfer does not result in integration of the transgene into the host genome (less than 0.1% adenovirus-mediated transfections result in transgene incorporation into host DNA), and therefore is not stable, adenoviral vectors can be propagated to high titer and allow gene transfer to non-replicating cells. Although the transgene is not passed to daughter cells, in the case of the adult cardiac myocytes, which do not divide, this is not an important limitation. Retroviral vectors provide stable gene transfer, and high titers are now obtainable via retrovirus pseudotyping ⁇ Burns, et al, Proc. Natl. Acad. Sci.
• Human 293 cells which are human embryonic kidney cells transformed with adenovirus EIA/EIB genes, typify the permissive cell lines.
• other cell lines which allow replication-deficient adenoviral vectors to propagate therein can be used.
• other cell lines useful for this purpose include HeLa cells.
• All adenoviral vectors used in the present invention can be constructed by the rescue recombination technique developed by Frank Graham ⁇ Virology, 163:614-617 (1988) ⁇ . Briefly, the transgene of interest is cloned into a shuttle vector that contains a promoter, polylinker and partial flanking adenoviral sequences from which EIA/EIB genes have been deleted.
• plasmid pACI (Virology, 163:614-617 (1988) ⁇ (or an analog) which encodes portions of the left end of the human adenovirus 5 genome ⁇ Virology, 163:614-617 (1988) ⁇ minus the early protein encoding E1A and E1B sequences that are essential for viral replication, and plasmid ACCMVPLPA ⁇ /. Biol. Chem., 267:25129-25134 (1992) ⁇ which contains polylinker, the CMV promoter and SV40 polyadenylation signal flanked by partial adenoviral sequences from which the EIA/EIB genes have been deleted can be exemplified.
• plasmid pACl or ACCMVPLA facilitates the cloning process.
• the shuttle vector is then co-transfected with a plasmid which contains the entire human adenoviral 5 genome with a length too large to be encapsidated, into 293 cells.
• Co-transfection can be conducted by calcium phosphate precipitation or lipofection under conditions such as those disclosed by Biotechniques 15:868-872 (1993).
• plasmid JM17 which encodes the entire human adenovirus 5 genome plus portions of the vector pBR322 including the gene for ampicillin resistance (4.3 kb) is exemplified.
• JM17 encodes all of the adenoviral proteins necessary to make mature viral particles, it is too large to be encapsidated (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 takes place so as to create a recombinant genome that is deficient in the EIA/EIB 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 encapsidated (see Figure 1).
• the initial mode of gene transfer uses adenoviral vectors as delineated above.
• the advantages of these vectors include the ability to effect high efficiency gene transfer (more than 60% of target organ cells transfected in vivo), the ease of obtaining high titer viral stocks and the ability of these vectors to effect gene transfer into cells such as cardiac myocytes which do not undergo rapid turnover.
• One potential disadvantage is that the current generation of this vector does not result in stable gene transfer. Genes transferred to the myocardium by adenovirus vectors do not integrate into the host cell DNA and, therefore, do not get passed on to the progeny of dividing cells (fibroblasts, endothelial cells, smooth muscle cells, etc.).
• Genes transferred to the myocardium by current generation adenoviral vectors remain active only for a period of weeks to months. This may actually be advantageous for certain clinical applications such as myocardial protection to induce a controlled amount of a stress related factor.
• newer generation adenoviral vectors that have further deletions in the adenovirus genome (in addition to EIA/EIB) are under development. These vectors have the potential to effect longer term gene transfer and to be less immunogenic. If it is determined that longer term gene transfer would be more efficacious and/or inflammatory response to first generation vectors becomes problematic, these newer generation vectors could be used.
• gene transfer limited to the arterial wall proves as efficacious as myocardial gene transfer to effect myocardial protection
• alternative method of gene transfer could be used including electroporation, use of hydrogel coated balloon catheters, use of liposomes or use of alternate viral vectors including retrovirus or adeno associated viral vectors.
• transgene expression is limited to ventricular cardiac myocytes.
• MLC_ y left ventricular myosin light chain-2
• the efficacy of gene expression and degree of specificity provided by the promoter with lacZ have been determined, using the recombinant adenoviral system of the present invention. Cardiac-specific expression has been documented previously by Lee, et al. ⁇ J. Biol.
• the MLC 2V promoter is comprised of 250 bp, and easily fits within the adenoviral-5 packaging constraints.
• the myosin heavy chain promcter known to be a vigorous promoter of transcription, cannot be used because its large size (5.5 kb) cannot fit within the adenoviral vector.
• the cardiac myocyte alone (that is without concomitant expression in endothelial cells, smooth muscle cells, and fibroblasts within the heart) will provide adequate expression of a stress related factor such as heat shock proteins HSP70i, HSP27, HSP40 and HSP60, and the adenosine A3 receptor to promote myocardial protection.
• a stress related factor such as heat shock proteins HSP70i, HSP27, HSP40 and HSP60
• Limiting expression to the cardiac myocyte also has advantages regarding the utility of gene transfer for the treatment of clinical myocardial ischemia. By limiting expression to the heart, one avoids any potentially harmful effect in non-cardiac tissues.
• the myocyte would likely provide the longest transgene expression since the cells do not undergo rapid turnover; expression would not therefore be decreased by cell division and death as would occur with endothelial cells. Subsequent studies will determine whether targeting gene expression to the endothelial cells, and limiting expression somewhat to the coronary endothelium by intracoronary injection, will be a sufficient means to deliver the transgene. Endothelial-specific promoters are already available for this purpose ⁇ Lee, et al., J. Biol. Chem., 265:10446-10450 (1990) ⁇ . As yet there are no fibroblast or smooth muscle cell promoters available that would efficiently limit expression of the transgene to smooth muscle or fibroblasts within the heart.
• Successful recombinant vectors can be plaque purified according to standard methods.
• the resulting viral vectors are propagated on 293 cells which provide E1A and E1B functions in trans to titers in the 10 10 -10 12 viral particles/ml range.
• Cells can be infected at 80% confluence and harvested at 36-48 hours post-infection. After 3 freeze-thaw cycles the cellular debris is pelleted by standard centrifugation and the virus further 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 resulting viral stock has a final viral titer in the range of 10 10 -10 12 viral particles/ml.
• the adenoviral construct must be highly purified, with no wild-type (potentially replicative) virus. Impure constructs can cause an intense immune response in the host animal. From this point of view, propagation and purification must be conducted to exclude contaminants and wild-type virus by, for example, identifying successful recombinants with PCR using appropriate primers, conducting two rounds of plaque purification, and double CsCl gradient ultracentrifugation.
• the viral stock can be in the form of an injectable preparation containing pharmaceutically acceptable carrier such as saline, as necessary.
• the final titer of the vector in the injectable preparation is preferably in the range of 10 10 -10 12 viral particles which allows for effective gene transfer.
• the adenovirus transgene constructs are delivered to the myocardium by direct intracoronary injection 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.
• the amount of the vector to be injected is preferably in the range of 10 10 -10 13 viral particles (more preferably 10 ⁇ -10 12 viral particles).
• the injection should be made deeply (such as 1 cm within the arterial lumen) into the lumen of the coronary arteries, and preferably be made in both coronary arteries, as the growth of collateral blood vessels is highly variable within individual patients.
• By injecting the material directly into the lumen of the coronary artery by coronary catheters it is possible to target the gene rather effectively, and to minimize loss of the recombinant vectors to the proximal aorta during injection. Gene expression when delivered in this manner is minimal in the liver, and viral RNA cannot be found in the urine at any time after intracoronary injection.
• Any variety of coronary catheter, or a Stack perfusion catheter, and so forth can be used in the present invention.
• the replication deficient recombinant adenoviral vectors of the present invention allow for highly efficient gene transfer in vivo without evidence for cytopathic effect or inflammation in the areas of gene delivery. Based on these results, it is believed that a high enough degree of in vivo gene transfer to effect in vivo functional changes is achieved. In particular, protective use of the vectors can be advantageous.
• stress proteins In order to provide optimal protection to the myocardium, stress proteins must be present at the time of ischemia. This requires gene transfer prior to anticipated ischemia. Although the timing of many prolonged ischemic episodes is unpredictable, there are specific settings during which ischemia is anticipated. These circumstances specifically allow for gene transfer prior to the ischemic event. The following include some of the clinical settings in which a role for a therapeutic gene transfer approach is anticipated:
• gene transfer could be effected by intracoronary injection of the viral construct several days prior to the planned non-cardiac surgery such that levels of protective stress factors in the myocardium would be high during the anticipated surgery.
• Cardiac catheterization, necessary for gene delivery, does not require anesthesia and is very well tolerated by otherwise clinically compromised patients.
• Percutaneous revascularization of the coronary vasculature is complicated 4% of the time by abrupt total closure of the target vessel.
• ischemia can often be aborted by use of intracoronary thrombolytic agents, placement of intracoronary stents or emergent bypass surgeries, frequently associated with irreversible myocardial damage.
• Even when abrupt vessel closure does not occur a significant number of procedures are complicated by "slow-flow" secondary to non- occlusive in situ thrombosis or micro-embolization to the distal coronary vasculature (common when treating diseased bypass grafts). These patients are also at high risk of peri-procedural myocardial damage.
• This subset includes patients with diffuse coronary disease in whom bypass surgery is technically not feasible, and patients with preclusive co- morbidity such as severe lung disease. In these patients, long-term gene transfer to protect the myocardium against chronic recurrent ischemia is believed to be particularly effective.
• Placement of an ameroid constrictor around the left circumflex (LCx) coronary artery results in gradual complete closure (within 7 days of placement) with minimal infarction (1% of the left ventricle, 4 ⁇ 1% of the LCx bed) ⁇ Roth, et al, Circulation, 82:1778 (1990), Roth, et al, Am. J. PhysioL, 235:H1279 (1987), White, et al, Circ. Res., 71:1490 (1992), Hammond, et al, Cardiol, 23:475 (1994), and Hammond, et al, J. Clin. Invest., 92:2644 (1993) ⁇ .
• ischemic region Myocardial function and blood flow are normal at rest in the region previously perfused by the occluded artery (referred to as the ischemic region), due to collateral vessel development, but blood flow reserve is insufficient to prevent ischemia when myocardial oxygen demands increase.
• the LCx bed is subject to episodic ischemia, analogous to clinical angina pectoris.
• Collateral vessel development and flow-function relationships are stable within 21 days of ameroid placement, and remain unchanged for four months ⁇ Roth, et al, Circulation, 82:1778 (1990), Roth, et al, Am. J. PhysioL, 235:H1279 (1987), White, et al, Circ. Res., 71:1490 (1992) ⁇ .
• the model has a bed with stable but inadequate collateral vessels, and is subject to periodic ischemia.
• Another distinct advantage of the model is that there is a normally perfused and functioning region (the LAD bed) adjacent to an abnormally perfused and functioning region (the LCx bed), thereby offering a "control" bed within each animal.
• Myocardial contrast echocardiography can be used to estimate regional myocardial perfusion in the present invention.
• the contrast material is composed of microaggregates of galactose and increases the echogenicity ("whiteness") of the image.
• the microaggregates distribute into the coronary arteries and myocardial walls in a manner that is proportional to blood flow ⁇ Skyba, et al, Circulation, 90:1513-1521 (1994) ⁇ .
• peak intensity of contrast is closely correlated with myocardial blood flow as measured by microspheres ⁇ Skyba, et al, Circulation, 90:1513-1521 (1994) ⁇ .
• a hydraulic cuff occluder can be placed around the proximal LCx adjacent to the ameroid.
• PCR can be used to detect stress related factor DNA and mRNA in myocardium from animals that has received gene transfer.
• myocardial samples from all five lacZ-infected animals show substantial ⁇ -galactosidase activity on histological inspection.
• using a polyclonal antibody to a stress related factor such as heat shock protein expressed in cells and in myocardium from animals that have received gene transfer can be demonstrated.
• 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 pLTR122E ⁇ Zhen, et al, Mol. Cell Biol, 8:3487 (1988) ⁇ as a 1.1 kb ECOR1 fragment which includes 981 bp of the open reading frame of the gene, and cloned into the polylinker of plasmid ACCMVPLPA which contains the CMV promoter and SV40 polyadenylation signal flanked by partial adenoviral sequences from which the ElA and ElB genes (essential for viral replication) had been deleted.
• This plasmid was co-transfected (lipofection) into 293 cells with plasmid JM17 which contained the entire human adenoviral 5 genome with an additional 4.3 kb insert making pJM17 too large to be encapsidated. Homologous rescue recombination resulted in adenoviral vectors containing the transgene in the absence of EIA/EIB sequences. Although these recombinants were nonreplicative in mammalian cells, they could propagate in 293 cells which had been transformed with EIA/EIB and provided these essential gene products in trans. Transfected cells were monitored for evidence of cytopathic effect which usually occurred 10-14 days after transfection.
• cell supernatant from plates showing a cytopathic effect was treated with proteinase K (50 mg/ml with 0.5% sodium dodecyl sulfate and 20 mM EDTA) at 56°C for 60 minutes, phenol/chloroform extracted and ethanol precipitated.
• Successful recombinants were then identified with PCR using primers ⁇ Biotechniques, 15:868-872 (1993) ⁇ complementary to the CMV promoter and SV40 polyadenylation sequences to amplify the insert (the expected 1.1 kb fragment), and primers ⁇ Biotechniques, 15:868-872 (1993) ⁇ designed to concomitantly amplify adenoviral sequences.
• Viral stocks were propagated in 293 cells to titers ranging between 10 10 and 10 12 viral particles, and were purified by double CsCl gradient centrifugation prior to use.
• Recombinant adenoviruses encoding j3-galactosidase, or HSP70i 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 10 - 10 12 viral particles range.
• Cells were infected at 80% confluence and harvested at 36-48 hours. After freeze-thaw cycles 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 -10 12 viral particles range.
• the adenoviral construct was highly purified, with no wild-type (potentially replicative) virus.
• rat cardiomyocytes were prepared by Langendorf perfusion with a collagenase containing perfusate according to standard methods. Rod shaped cells were cultured on laminin coated plates and at 24 hours were infected with the 3-galactosidase-encoding adenovirus obtained in the above Experiment 1 at a multiplicity of infection of 1:1. After a further 36 hour period the cells were fixed with glutaraldehyde and incubated with X-gal. Consistently 70-90% of adult myocytes expressed the 3-galactosidase transgene after infection with the recombinant adenovirus. At a multiplicity of infection of 1-2:1 there was no cytotoxicity observed.
• EXPERIMENT 3 Pig Myocardium In Vivo
• the ⁇ -galactosidase-encoding adenoviral vector obtained in the above Experiment 1 was propagated in permissive 293 cells and purified by CsCl gradient ultracentrifugation with a final viral titer of 1.5 x 10 10 viral particles, based on the procedures of Experiment 1.
• An anesthetized, ventilated 40 kg pig underwent thoracotomy and isolation of the left circumflex and left anterior descending coronary arteries.
• a 26 gauge butterfly needle was inserted in the mid left anterior descending (LAD) coronary artery and the vector (1.5 x 10 10 viral particles) was injected in a 2 ml volume.
• the chest was closed and the animal allowed to recover.
• the animal was sacrificed.
• Animals includes 14 domestic pigs, (30-40 kg).
• a left thoracotomy is performed under sterile conditions for instrumentation.
• Catheters are placed in the left atrium and aorta, providing a means to measure regional blood flow, and to monitor pressures. Wires are sutured on the left atrium to permit ECG recording and atrial pacing.
• an ameroid is placed around the proximal LCx.
• this treatment group receives an adenoviral construct that includes genes for HSP70i (a heat shock protein), driven by a CMV promoter.
• Control animals receives gene transfer with an adenoviral construct that includes a reporter gene, lacZ, driven by a CMV promoter.
• the helper independent replication deficient human adenovirus 5 system constructed in the above Experiment 1 is used.
• the genes of interest are lacZ and hsp70i.
• the material injected in vivo is highly purified and contains no wild-type (replication competent) adenovirus.
• the possible in vivo adenoviral infection and inflammatory infiltration in the heart are minimized.
• By injecting the material directly into the lumen of the coronary artery by coronary catheters it is possible to "target" the gene rather effectively. Gene expression when delivered in this manner in minimal in the liver, and viral RNA cannot be found in the urine at any time after intracoronary injection.
• Animals are anesthetized, and arterial access acquires via the right carotid by cut-down; a 5F Cordis sheath is placed.
• a 5F Multipurpose (A2) coronary catheter is used to engage the coronary arteries. Closure of the LCx ameroid is confirmed by contrast injection into the left main coronary artery. The catheter tip is then placed 1 cm within the arterial lumen so that minimal material will be lost to the proximal aorta during injection. This procedure is carried out for each of the pigs.
• the strategy for myocardial protective studies include the timing of transgene delivery, the route of administration of the transgene, and choice of the stress related gene, using the aforesaid construct including a reporter gene (lacZ) and that including a stress related factor gene as well as the aforesaid pig models.
• the ameroid model of myocardial ischemia is chosen, and gene transfer is performed after stable. Gene transfer are effected by intracoronary injection of the viral construct several days prior to non-cardiac surgery or a diagnostic cardiac catheterization such that levels of protective stress factors in the myocardium will be high during the anticipated surgery or percutaneous revascularization. In addition, gene transfer by intracoronary injection is conducted at the time of diagnostic cardiac catheterization just prior to surgery. Myocardial protection can be assessed by the aforesaid echocardiography and microscopic analysis.
• Neonatal rat cardiomyocytes were cultured as previously described ⁇ Iwaki, et al, Circulation, 87:2023-2032 (1993) ⁇ .
• the embryonic rat heart-derived cell line H9c2(2-1) and the human embryonic kidney cell line 293 were both obtained from the American Type Culture Collection, Rockville, MD, and were maintained in DMEM supplemented with antibiotics (penicillin/streptomycin/fungizone) and 10% fetal calf serum (FCS).
• FCS fetal calf serum
• MOI multiplicity of infection
• cells were incubated with viral constructs for 60 minutes with mild constant shaking; 2 ml of DMEM/2% heat-inactivated FCS was then added and the plates incubated for 2 days in a 37°C, C0 2 incubator. Simulated ischemia of the infected neonatal rat cardiomyocytes and H9c2 plates were done as previously described ⁇ Mestril, et al, J. Clin. Invest., 98:759-767 (1994), hereby incorporated by reference in its entirety) ⁇ .
• the rat hsp70 gene was cloned into the multiple cloning site of the adenoviral shuttle plasmid pACCMVpLpASR- (kindly provided by Dr. Robert D. Gerard, University of Texas, Southwestern Medical Center) ⁇ Gomez-Fox, et al, J. Biol. Chem., 267:25129-25134 (1992) ⁇ .
• This plasmid contains the 5' end of the adenovirus serotype 5 genome (map units 0 to 17) where the El region has been substituted with the human cytomegalovirus enhancer-promoter followed by the multiple cloning site from pACl9 and the polyadenylation region from SV40.
• the resulting plasmid was co-transfected with pJM17, a plasmid that contains the complete adenovirus 5 genome, into the human embryonic kidney cell line 293 using the calcium phosphate transfection method. Infectious viral particles containing the inserted hsp70 were generated by in vivo recombination in the 293 cells and were isolated as single plaques seven days later.
• adenoviral construct that consisted of the pACCMVpLASR- plasmid without any insert.
• the isolated plaques were propagated in 293 cells for several passages to obtain high titer stocks.
• Viral particles were purified by CsCl ultracentrifugation.
• the titer of viral stocks was determined either by plaque assay or deproteination of an aliquot of the viral stock and amount of DNA determined by optical density ⁇ Barr, et al, Gene Therapy, 1:51-58 (1994) ⁇ .
• Cellular protein extracts were prepared from neonatal cardiomyocytes and H9c2 cells infected with adenoviral-/ «p70/, the control adenoviral-SR- constructs or non-infected as previously described ⁇ Mestril, et al, J. Clin. Invest., 93:759-767 (1994) ⁇ . Protein concentration was determined by the Bradford Assay (BioRad Laboratories, Richmond, CA). Protein samples (40 ⁇ g each) were fractionated on an 8% SDS-polyacrylamide gel and electrotransferred onto nitrocellulose using a semi-dry electrotransfer apparatus (BioRad Laboratories).
• nitrocellulose blots were reacted either with a monoclonal antibody C92F3A-5 (StressGen, Biotechnologies Corp., Victoria, BC) which binds specifically to the mammalian inducible HSP70 or with a polyclonal antiserum which binds to the COOH terminal of the mammalian HSP70s and HSP90s ⁇ Mehta, et al, Circ. Res., 63:512-517 (1988) ⁇ .
• C92F3A-5 StressGen, Biotechnologies Corp., Victoria, BC
• Blots were subsequently reacted with biotinylated secondary antibodies and streptavidin- horseradish peroxidase-conjugated systems (Vectastain, ABC kit; Vector Laboratories, Burlingame, CA) and developed with diaminobenzidine, tetrahydrochloride (DAB kit, Vector Laboratories).
• Indirect Immunofluorescence Plates of infected and non-infected neonatal cardiomyocytes and H9c2 cells were washed twice with ice cold PBS and fixed with 100% ice cold methanol for 2 minutes. The fixed cells were then rehydrated with TBS containing 0.1% bovine serum albumin and reacted either with the monoclonal antibody against the inducible HSP70 (C92F3A-5) and subsequently developed with an ABC kit and VectorRed kit (Vector Laboratories) or a FITC-conjugated polyclonal antibody raised against the hexon coat protein of adenovirus (AB1056F, Chemicon International, Temecula, CA).
• Creatine kinase (CK) activity was measured spectrophotometrically using a commercial CK kit (Sigma Immunochemicals, St.Louis, MO). CK activity release was expressed as the percent of the total CK activity present in each plate normalized by the amount of protein in each plate.
• Lactate dehydrogenase (LDH) activity was determined spectrophotometrically using a LDH test kit (Sigma). LDH activity released was expressed as the percent of the total LDH present in each plate normalized by the amount of protein present.
• Results are expressed as the mean +. standard error. Statistical significance was assessed by the Student's two-tailed test, unpaired t test and a probability value of ⁇ 0.05 was considered significant.
• a replication-deficient recombinant adenoviral vector containing the inducible rat hsp70 gene ⁇ Mestril, et al. , Biochem. J. , 298:561-569 (1994) ⁇ The general strategy used to introduce a foreign gene into the El region of the replication-deficient adenoviral vector is represented schematically in Figure 2 (see also Materials and Methods).
• the control adenoviral construct was generated using the same scheme with the exception that it lacks an insert.
• protein extracts were prepared from neonatal rat cardiomyocytes 48 hours after infection. The protein extracts were examined by Western blot analysis. During the course of this study, three Western blots produced identical results.
• a representative Western blot was developed with a polyclonal antibody that binds to both HSP70 and HSP90.
• the Western blot has three lanes. The first lane contained proteins from non- infected myocytes. The second lane contained proteins from myocytes infected with the control adenoviral vector (adenoviral-SR) at a MOI of 10:1.
• the third lane contained proteins from myocytes infected with the adenoviral- wp70 (MOI of 10:1).
• the Western blot showed that the adenoviral- up70z construct infected myocytes constitutively expressed a large amount of the exogenous hsp70i.
• the second Western blot showed that while at a MOI of 1:1, the level of expression of HSP70 obtained with the adenoviral- w ⁇ 70 was lower than at a MOI of 10:1, it was still comparable to the normal expression of hsp70i in non-infected heat shocked cardiomyocytes (42°C, 60 minutes).
• control adenoviral vector (adenoviral-SR) lacked an insert
• indirect immunofluorescence was used to detect infection by this adenoviral construct as well as that of the adenoviral- wp70/ construct in neonatal myocytes and H9c2 cells by using a polyclonal antibody that binds to the hexon assembly protein of adenovirus. The result was obtained of such an analysis on H9c2 cells that were infected with the adenoviral constructs 48 hours prior to fixation of the cells.
• Panels A and B of the indirect immunofluorescence were infected with the adenoviral-/wp70/ construct (MOI of 1:1), panels C and D were infected with the control adenoviral-SR construct (MOI of 1:1) and panels E and F were non-infected cells.
• panels A, C and E cells were reacted with the monoclonal antibody against the inducible HSP70.
• panels B, D and F cells were reacted with the polyclonal antibody against the adenoviral hexon assembly protein.
• High levels of expression of hsp70i could only be observed in cells infected with the adenoviral-/up70z and reacted with the monoclonal antibody specific to the HSP70i (panel A).
• H9c2 cells were infected either with the adenoviral-/wp70i (designated "Adhsp70” in Figure 3) (MOI of 1:1) or the adenoviral-SR (designated "AdSR-” in Figure 3) (MOI of 1:1), and 48 hours later these cells were submitted to simulated ischemia. Applicants then measured the amount of lactate dehydrogenase activity released and remaining after simulated ischemia as a parameter of cellular damage. Figure 3 shows the results obtained from six independent experiments.
• lactate dehydrogenase (LDH) released is expressed as a percentage of LDH released in control plates (infected but not submitted to simulated ischemia) which is taken as 100%.
• the amount of LDH released was calculated as the amount of LDH activity released, normalized by the amount of protein released (Units/mg) over the amount of total LDH activity normalized by the total amount of protein in each plate (total Units/mg).
• the p value is less that 0.05, indicating a statistically significant difference, and denoted by the "*" in Figure 3.
• the amount of CK released was calculated as the amount of CK activity released, normalized by the amount of protein released (Units/mg) over the amount of total CK activity, normalized by the total amount of protein in each plate (total Units/mg).
• the p value is less than 0.05, indicating a statistically significant difference, and denoted by the "*" in Figure 4.
• the experiment supports the introduction of adenoviral constructs of the present invention into the hearts of animals to confer protection against myocardial ischemia.

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