Claims
- 1. An isolated and purified recombinant adenoviral vector, said vector comprising:
an adenoviral genome from which the E1A/E1B genes have been deleted; a transgene coding for a stress related factor which is a heat shock protein or the adenosine A3 receptor; and a promoter operably linked to said transgene, wherein expression of the transgene is controlled by said promoter.
- 2. The vector of claim 1, wherein said stress related factor is selected from the group consisting of HSP70i, HSP27, HSP40, HSP60, and the adenosine A3 receptor.
- 3. The vector of claim 1, wherein said promoter is a CMV promoter.
- 4. The vector of claim 1, wherein said promoter is a ventricular myocyte-specific promoter.
- 5. A method of producing an isolated and purified recombinant vector of claim 1, comprising the steps of:
cloning a transgene coding for a stress related factor into a plasmid containing a promoter and a polylinker flanked by 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 plasmid having the entire adenoviral genome so as to create a recombinant genome containing the transgene without the E1A/E1B genes, said recombinant genome being sufficiently small to be encapsulated; identifying cells comprising recombinant vectors in cell cultures; propagating the resulting recombinant vectors in mammalian cells transformed with the E1A/E1B genes; and purifying the propagated recombinant vectors.
- 6. The method of claim 5, wherein said plasmid into which the transgene is cloned is plasmid pAC1 or plasmid ACCMVPLPA.
- 7. The method of claim 5, wherein said identification comprises the steps of:
monitoring transfected cells for evidence of cytopathic effect; treating the cell supernatant from cell cultures showing a cytopathic effect with proteinase K, followed by phenol/chloroform extraction and ethanol precipitation to isolate viral DNA; identifying cells producing recombinant vectors with PCR using primers complementary to the CMV promoter and primers complementary to adenoviral sequences; and purifying recombinant vectors using two rounds of plaque purification.
- 8. The method of claim 5, wherein said purification comprises the steps of:
propagating the resulting recombinant vectors in cells transformed with the E1A/E1B genes to titers in the 1010-1012 viral particles range; purifying the propagated recombinant vectors by double CsCl gradient ultracentrifugation; and filtering the purified recombinant vectors through sepharose columns.
- 9. The method of claim 5, wherein said stress related factor is selected from the group consisting of HSP70i, HSP27, HSP40, HSP60, and the adenosine A3 receptor.
- 10. A method of elevating the level of stress related factor in the myocardium of a patient, comprising delivering a replication-deficient viral vector to the myocardium of a patient, wherein said vector comprises a transgene encoding a stress related factor, and wherein delivery is by intracoronary injection into the lumen of one or both coronary arteries of said patient.
- 11. The method of claim 10, wherein the stress related factor is a heat shock protein or the adenosine A3 receptor.
- 12. The method of claim 10, wherein said stress related factor is selected from the group consisting of HSP70i, HSP27, HSP40, HSP60, and the adenosine A3 receptor.
- 13. The method of claim 10, wherein the stress related factor is a heat shock protein or the adenosine A3 receptor, and wherein the vector is an adenoviral vector comprising a gene encoding said heat shock protein or the adenosine A3 receptor.
- 14. The method of claim 10, wherein said patient has non-revascularized ischemic heart disease and wherein said vector is administered a plurality of days prior to non-cardiac surgery.
- 15. The method of claim 10, wherein said vector is delivered at the time of a diagnostic catheterization a plurality of days prior to complex percutaneous revascularization.
- 16. The method of claim 10, wherein said vector is delivered at the time of a diagnostic cardiac catheterization.
- 17. The method of claim 10, wherein said vector is delivered at the time of a diagnostic coronary angiography.
- 18. The method of claim 10, wherein said promoter is a CMV promoter.
- 19. The method of claim 10, wherein said promoter is a ventricular myocyte-specific promoter.
- 20. The method of claim 10, wherein said vector is delivered in the form of a viral stock having a final viral titer of 1010-1013 viral particles.
Government Interests
[0001] This invention was made with Government support under Grant Nos. R01 HL-49343 and K14 HL-03150, awarded by the National Institute of Health. The Government has certain rights in this invention.
Divisions (1)
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10741907 |
Dec 2003 |
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Continuations (2)
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Continuation in Parts (2)
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