Viral (and other) techniques in gene therapy for hypertension

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