Viral%20(and%20other)%20techniques%20in%20gene%20therapy%20for%20hypertension

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Viral (and other) techniques in gene therapy for
hypertension

• Justin Grobe
• Oral Qualifying Exam
• and
• Dissertation Work Proposal

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Hypertension

• 50 million (1 in 5) Americans age 6 and older
  have high blood pressure (gt 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.
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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.
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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.
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Current therapies for hypertension

• Renin-Angiotensin System Interference
• Angiotensin Converting Enzyme (ACE) inhibitors
  Captopril, Enalapril
• Angiotensin II (type 1) receptor blockers
  (ARBs) Losartan

Stringer, J. L. Basic Concepts in Pharmacology,
2nd ed. McGraw-Hill Medical Publishing Division,
New York. 2001.
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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.
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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

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Genetic therapy delivery methods

• Physical
• Molecular (Non-viral)
• Viral

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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)

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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)

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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

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Common virus types for gene therapy

• Adenovirus
• Adeno-associated viruses (AAV)
• Retroviruses
• Lentiviruses
• Helper-dependent AAV

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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)

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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!)

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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)

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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)

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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

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Practical Challenges with Viruses

• Safety
• Toxicity
• Immune reactions
• Integration Position and genomic effects
• Efficacy
• Control of transgene expression

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Ethical Challenges

• Questionable need, considering the risks?
• Regulation of transgene?
• Population genetics and eugenics?

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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
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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
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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

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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

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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

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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

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

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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
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Two vectors in vitro
(Detection Limit)
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Two vectors in vivosystemic 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?

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Two vectors in vivoplans 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?)

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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

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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