Viral (and other) techniques in gene therapy for hypertension

Justin GrobeOral Qualifying Exam

andDissertation Work Proposal

Hypertension50 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 idiopathicEducation 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.

Current therapies for hypertensionDiuretics

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.

Current therapies for hypertensionPeripheral 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.

Current therapies for hypertensionRenin-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.

Problems with conventional methodsOf those with hypertension,

31.6% are unaware27.4% are on medication and have it controlled26.2% are on medication but do not have it controlled14.8% are aware but are not on medication

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.

Issues of complianceCost, availability, understanding

Unaware

Medicated,Controlled

Medicated,Not Controlled

Aware,No Meds

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

Genetic therapy delivery methodsPhysical

“Molecular” (Non-viral)

Viral

Physical methods“Gene-gun” method

Used for plant research (only!)Plasmid-coated superfine beads fired from a .22 caliber chamberHighly inaccurate and inefficient (kills most cells)

Non-viral, “molecular” methodsLiposomes and naked DNAElectroporation methodSalt-shock methods (CaCl2)

Harsh, non-specific, (usually transient), can be inefficient

Agrobacterium tumefaciens “Ti-plasmid” methodUsed in plants (dicots only)

Viral methodsMany virus types available with varying:

Target specifictyDividing/Non-dividing cellsCassette sizeTransfection stabilityGenome insertion areasGerm-line/Somatic cellsEfficiency

Common virus types for gene therapy

AdenovirusAdeno-associated viruses (“AAV”)RetrovirusesLentivirusesHelper-dependent AAV

AdenovirusNon-enveloped, linear ds-DNAInfect dividing and non-dividing cells (good)High titers possible during production (good)Do not integrate into host genome well (bad)

The Adeno-Associated VirusSmall ss-DNANot 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!)

RetrovirusRNA, depend on viral enzymesIntegrates 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)

LentivirusSub-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)

Helper-dependent AAV

Very newVery secret (patent restrictions)Most of the same characteristics as AAV, except;HUGE PAYLOAD CASSETE SIZE - 30 to 60 kb

Practical Challenges with VirusesSafety

ToxicityImmune reactionsIntegration – Position and genomic effects

EfficacyControl of transgene expression

Ethical ChallengesQuestionable need, considering the risks?Regulation of transgene?Population genetics and eugenics?

Practical and Ethical Challenge: Transgene Control

One approach: tetracycline-regulatable systemsTet-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

rTA

Tet-operator PromoterTransgene of

interest

(Without tetracycline)

(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

rtTA

Tet-operator PromoterTransgene of

interest

(With tetracycline)

(Without tetracycline)

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

Together:Hypertension therapy needs a new directionGene therapy may be that directionThe 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 controlThe tetracycline-regulatable systems are examples of such transcriptional control systems

Research hypothesisAn 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

Regulating gene therapy for hypertension: proposed project plan

Clone tet-system and therapeutic genesProduce viruses containing system Establish transgene control with reporter genes

In vitroIn vivo

Induce therapeutic genesReverse hypertension in vivoReverse end-organ damage in vivo

Hypertension target genes: the RAS

Angiotensinogen

Angiotensin II

Angiotensin I

AT1R AT2R

Renin

ACE, Chymase

tPA Angiotensin (1-9)

Angiotensin (1-7)

Mas / (AT1-7R?)

ACE

ACE2

ACE2

Endopeptidases

Hypertension target genes: Angiotensinogen

Angiotensinogen

Angiotensin (1-9)

Angiotensin II

Angiotensin I

Angiotensin (1-7)

AT1R AT2R Mas / (AT1-7R?)

Renin

ACE, Chymase ACE

ACE2

ACE2

tPA

Endopeptidases

Hypertension target genes: ACE2

Angiotensinogen

Angiotensin (1-9)

Angiotensin II

Angiotensin I

Angiotensin (1-7)

AT1R AT2R Mas / (AT1-7R?)

Renin

ACE, Chymase ACE

ACE2

ACE2

tPA

Endopeptidases

ReporterViral Constructs: single vector

EF1a - elongation factor 1 alphartTA - “Tet-ON”IRES - internal ribosome entry sitetTS - tet-silencerTRE - tetracycline responsive elementPLAP - placental alkaline phosphatase

rtTA IRES tTSEF1a PLAPTRE poly A

(+ Dox)

Single vector effectsIn vitro titer:

No virus - 0 cells/mLVirus, no Dox - 1.98x106

Virus, Dox - 1.15x107 (6x induction)

In vivo staining:No staining in heart, liver, lung of any animal

ReporterViral Constructs: two vectors

EF1a - elongation factor 1 alphartTA - “Tet-ON”IRES - internal ribosome entry sitetTS - tet-silencerTRE - tetracycline responsive elementSEAP - secreted alkaline phosphatase

SEAPTRE poly A

rtTA IRES tTSEF1a poly A

(+ Dox)

Two vectors in vitro

60

70

80

90

100

110

120

130

140

TRE-SEAP EF1a-rtTA-IRES-tTSand TRE-SEAP

Lu

min

esce

nce

(R

LU

)

No Doxycycline

(1 ug/uL) Doxycycline

(Detection Limit)

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 daysSubcutaneous injection, ad. lib. in drinking water

ProblemsNo positive control group - assay?Systemic delivery & simple probability - design?

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 injectionACE2 - any tissue (skeletal muscle?)

Current workRT-PCR of systemic two-vector animal tissues (heart, liver) to measure rtTA and SEAP transcriptsCloning positive control for SEAP (EF1a - SEAP)Working on making transgenic rat which expresses rtTA and tTS proteins constituitively and ubiquitouslyProducing three viruses

EF1a-SEAPEF1a-rtTA-IRES-tTSTRE-SEAP

Future plansIn vivo reporter gene experiment with local delivery and positive control groupClone therapeutic gene into TYF-TRE plasmid (“second vector”)Produce virusesIn vivo blood pressure and end-organ damage experiments

HypertrophyVascular Reactivity