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Viral (and other) techniques in gene therapy for hypertension

Viral (and other) techniques in gene therapy for hypertension. Justin Grobe Oral Qualifying Exam and Dissertation Work Proposal. Hypertension. 50 million (1 in 5) Americans age 6 and older have high blood pressure (> 140/90 mmHg) and/or are taking antihypertensive medicine

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Viral (and other) techniques in gene therapy for hypertension

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  1. Viral (and other) techniques in gene therapy for hypertension Justin Grobe Oral Qualifying Exam and Dissertation Work Proposal

  2. Hypertension • 50 million (1 in 5) Americans age 6 and older have high blood pressure (> 140/90 mmHg) and/or are taking antihypertensive medicine • 90-95% of primary hypertension cases are idiopathic • Education and income levels are negatively correlated with blood pressure (affordability of treatment?) American Heart Association. 2002 Heart and Stroke Statistical Update. Dallas, TX: American Heart Association, 2001.

  3. Current therapies for hypertension • Diuretics • Thiazide Diuretics [Chlorothiazide, Hydrochlorothiazide] • Loop Diuretics [Furosemide] • Potassium-Sparing Diuretics [Spironolactone] Stringer, J. L. Basic Concepts in Pharmacology, 2nd ed. McGraw-Hill Medical Publishing Division, New York. 2001.

  4. Current therapies for hypertension • Peripheral Resistance Reducers • Direct Vasodilators • Calcium Channel Blockers [Diltiazem, Nifedipine, Verapamil] • Nitrates [Nitroglycerin, Nitroprusside] • Others [Hydralazine, Minoxidil] • Sympathetic Nervous System Depressants • Alpha-1 Blockers [Prazosin] • Beta-(1 and 2) Blockers [Propranolol] • Alpha-2 Agonists [Clonidine] Stringer, J. L. Basic Concepts in Pharmacology, 2nd ed. McGraw-Hill Medical Publishing Division, New York. 2001.

  5. Current therapies for hypertension • Renin-Angiotensin System Interference • Angiotensin Converting Enzyme (ACE) inhibitors [Captopril, Enalapril] • Angiotensin II (type 1) receptor blockers (“ARB’s”) [Losartan] Stringer, J. L. Basic Concepts in Pharmacology, 2nd ed. McGraw-Hill Medical Publishing Division, New York. 2001.

  6. Problems with conventional methods • Of those with hypertension, • 31.6% are unaware • 27.4% are on medication and have it controlled • 26.2% are on medication but do not have it controlled • 14.8% are aware but are not on medication Aware, No Meds Unaware Medicated, Not Controlled Medicated, Controlled • Issues of compliance • Cost, availability, understanding American Heart Association. 2002 Heart and Stroke Statistical Update. Dallas, TX: American Heart Association, 2001. JM Mallion, D Schmitt. Patient complaince in the treatment of arterial hypertension. Journal of Hypertension. 19(12): 2281-2283. 2001.

  7. Potential solution: Gene therapy • Ideally, • Single treatment, once in lifetime of patient (a “cure”) • 100% compliance, since no behavior is required • Cost / Availability would favor treatment for poor and/or uneducated individuals by their health care providers

  8. Genetic therapy delivery methods • Physical • “Molecular” (Non-viral) • Viral

  9. Physical methods • “Gene-gun” method • Used for plant research (only!) • Plasmid-coated superfine beads fired from a .22 caliber chamber • Highly inaccurate and inefficient (kills most cells)

  10. Non-viral, “molecular” methods • Liposomes and naked DNA • Electroporation method • Salt-shock methods (CaCl2) Harsh, non-specific, (usually transient), can be inefficient • Agrobacterium tumefaciens “Ti-plasmid” method • Used in plants (dicots only)

  11. Viral methods • Many virus types available with varying: • Target specificty • Dividing/Non-dividing cells • Cassette size • Transfection stability • Genome insertion areas • Germ-line/Somatic cells • Efficiency

  12. Common virus types for gene therapy • Adenovirus • Adeno-associated viruses (“AAV”) • Retroviruses • Lentiviruses • Helper-dependent AAV

  13. Adenovirus • Non-enveloped, linear ds-DNA • Infect dividing and non-dividing cells (good) • High titers possible during production (good) • Do not integrate into host genome well (bad)

  14. The Adeno-Associated Virus • Small ss-DNA • Not much immune response (very good!) • Infects both dividing and non-dividing cells (good) • Somewhat difficult to produce at high titers (bad) • Very small cassette – 3 kb (bad?) • Integration into host genome specifically into an “unimportant” portion of chromosome 19 (very very good!)

  15. Retrovirus • RNA, depend on viral enzymes • Integrates into genome (good), but in very random positions (potentially very bad – cancer!) • Only infects dividing cells (bad?) • Difficult to obtain high titers in production (bad), but easy to make large volumes (good) • Large cassette sizes possible (very good)

  16. Lentivirus • Sub-family of retroviruses (HIV family) • Same traits of retroviruses, EXCEPT: • Ability to transduce non-dividing cells (very good!) • High titers possible in production (good) • Large scale production yields small volume (bad) • Animal care and use issues (because of HIV origins)

  17. Helper-dependent AAV • Very new • Very secret (patent restrictions) • Most of the same characteristics as AAV, except; • HUGE PAYLOAD CASSETE SIZE - 30 to 60 kb

  18. Practical Challenges with Viruses • Safety • Toxicity • Immune reactions • Integration – Position and genomic effects • Efficacy • Control of transgene expression

  19. Ethical Challenges • Questionable need, considering the risks? • Regulation of transgene? • Population genetics and eugenics?

  20. (With tetracycline) rTA (Without tetracycline) Practical and Ethical Challenge: Transgene Control • One approach: tetracycline-regulatable systems • Tet-OFF (rTA) • Constitutive rTA protein expression (blocks transcription) • Presence of a tetracycline (doxycycline has low side-effects) causes release of the rTA suppressive protein from the tet-operator, allows transcription of transgene Strong promoter (tissue specific?) rTA Tet-operator Promoter Transgene of interest

  21. (Without tetracycline) rtTA (With tetracycline) Practical and Ethical Challenge: Transgene Control • Tet-ON (rtTA) • Constitutive rtTA protein expression (transcription factor) • Presence of tetracycline causes binding of rtTA to operator, inducing transcription • Small amout of leak usually observed in absence of tetracyclines Strong promoter (tissue specific?) rtTA Tet-operator Promoter Transgene of interest

  22. Practical and Ethical Challenge: Transgene Control • New generations of the tetracycline-regulatable systems incorporate both tet-ON and tet-OFF, and new tet-Silencer sequences • Even tighter control over transgene • “Off” is really off

  23. Together: • Hypertension therapy needs a new direction • Gene therapy may be that direction • The lentiviruses allow large transgene cassettes to be stably transfected in vivo • Larger cassette sizes allow for incorporation of transcriptional control systems, overcoming the practical and ethical dilemma of transgene control • The tetracycline-regulatable systems are examples of such transcriptional control systems

  24. Research hypothesis • An anti-hypertensive therapeutic gene, delivered via a Lenti-based viral vector, and under the control of a tetracycline-sensitive promoter system, will alleviate hypertension and reverse hypertension-associated end-organ damage in a regulatable manner

  25. Regulating gene therapy for hypertension: proposed project plan • Clone tet-system and therapeutic genes • Produce viruses containing system • Establish transgene control with reporter genes • In vitro • In vivo • Induce therapeutic genes • Reverse hypertension in vivo • Reverse end-organ damage in vivo

  26. Angiotensinogen Renin ACE2 tPA Angiotensin I Angiotensin (1-9) Endopeptidases ACE, Chymase ACE ACE2 Angiotensin II Angiotensin (1-7) AT1R AT2R Mas / (AT1-7R?) Hypertension target genes: the RAS

  27. Hypertension target genes: Angiotensinogen Angiotensinogen Renin ACE2 tPA Angiotensin I Angiotensin (1-9) Endopeptidases ACE, Chymase ACE ACE2 Angiotensin II Angiotensin (1-7) AT1R AT2R Mas / (AT1-7R?)

  28. Hypertension target genes: ACE2 Angiotensinogen Renin ACE2 tPA Angiotensin I Angiotensin (1-9) Endopeptidases ACE, Chymase ACE ACE2 Angiotensin II Angiotensin (1-7) AT1R AT2R Mas / (AT1-7R?)

  29. (+ Dox) EF1a TRE rtTA IRES tTS PLAP poly A ReporterViral Constructs: single vector EF1a - elongation factor 1 alpha rtTA - “Tet-ON” IRES - internal ribosome entry site tTS - tet-silencer TRE - tetracycline responsive element PLAP - placental alkaline phosphatase

  30. Single vector effects • In vitro titer: • No virus - 0 cells/mL • Virus, no Dox - 1.98x106 • Virus, Dox - 1.15x107 (6x induction) • In vivo staining: • No staining in heart, liver, lung of any animal

  31. EF1a rtTA IRES tTS poly A (+ Dox) TRE SEAP poly A ReporterViral Constructs: two vectors EF1a - elongation factor 1 alpha rtTA - “Tet-ON” IRES - internal ribosome entry site tTS - tet-silencer TRE - tetracycline responsive element SEAP - secreted alkaline phosphatase

  32. Two vectors in vitro (Detection Limit)

  33. Two vectors in vivo:systemic delivery • No SEAP detected in blood of animals with or without doxycycline-induction • Basal, 2 days, 7 days, 12 days, 17 days • Subcutaneous injection, ad. lib. in drinking water • Problems • No positive control group - assay? • Systemic delivery & simple probability - design?

  34. Two vectors in vivo:plans for local delivery • To increase probability of infection by both vectors in same target cells, reduce total number of target cells • Antisense to angiotensinogen - hepatic-portal injection • ACE2 - any tissue (skeletal muscle?)

  35. Current work • RT-PCR of systemic two-vector animal tissues (heart, liver) to measure rtTA and SEAP transcripts • Cloning positive control for SEAP (EF1a - SEAP) • Working on making transgenic rat which expresses rtTA and tTS proteins constituitively and ubiquitously • Producing three viruses • EF1a-SEAP • EF1a-rtTA-IRES-tTS • TRE-SEAP

  36. Future plans • In vivo reporter gene experiment with local delivery and positive control group • Clone therapeutic gene into TYF-TRE plasmid (“second vector”) • Produce viruses • In vivo blood pressure and end-organ damage experiments • Hypertrophy • Vascular Reactivity

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