Download GROBE_ORAL_EXAM

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
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.
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.
Current therapies for hypertension
Peripheral Resistance Reducers
Direct Vasodilators
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Calcium Channel Blockers [Diltiazem, Nifedipine, Verapamil]
Nitrates [Nitroglycerin, Nitroprusside]
Others [Hydralazine, Minoxidil]
Sympathetic Nervous System Depressants
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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 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.
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.
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 methods
Physical
“Molecular” (Non-viral)
Viral
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)
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)
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
Common virus types for gene therapy
Adenovirus
Adeno-associated viruses (“AAV”)
Retroviruses
Lentiviruses
Helper-dependent AAV
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)
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!)
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)
Lentivirus
Sub-family of retroviruses (HIV family)
Same traits of retroviruses, EXCEPT:
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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 new
Very secret (patent restrictions)
Most of the same characteristics as AAV, except;
HUGE PAYLOAD CASSETE SIZE - 30 to 60 kb
Practical Challenges with Viruses
Safety
Toxicity
Immune reactions
Integration – Position and genomic effects
Efficacy
Control of transgene expression
Ethical Challenges
Questionable need, considering the risks?
Regulation of transgene?
Population genetics and eugenics?
Practical and Ethical Challenge:
Transgene Control
One approach: tetracycline-regulatable systems
Tet-OFF (rTA)
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Constitutive rTA protein expression (blocks transcription)
Presence of a tetracycline (doxycycline has low sideeffects) causes release of the rTA suppressive protein
from the tet-operator, allows transcription of transgene
(With tetracycline)
rTA
(Without tetracycline)
Strong promoter
(tissue specific?)
rTA
Tet-operator
Promoter
Transgene of
interest
Practical and Ethical Challenge:
Transgene Control
Tet-ON (rtTA)

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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
(Without tetracycline)
rtTA
(With tetracycline)
Strong promoter
(tissue specific?)
rtTA
Tet-operator
Promoter
Transgene of
interest
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 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
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
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
Hypertension target genes: the RAS
Angiotensinogen
Renin
tPA
Angiotensin I
ACE2
ACE, Chymase
Angiotensin II
AT1R
AT2R
ACE2
Angiotensin (1-9)
ACE
Angiotensin (1-7)
Mas / (AT1-7R?)
Hypertension target genes: Angiotensinogen
Angiotensinogen
Renin
tPA
Angiotensin I
ACE2
ACE, Chymase
Angiotensin II
AT1R
AT2R
ACE2
Angiotensin (1-9)
ACE
Angiotensin (1-7)
Mas / (AT1-7R?)
Hypertension target genes: ACE2
Angiotensinogen
Renin
tPA
Angiotensin I
ACE2
ACE, Chymase
Angiotensin II
AT1R
AT2R
ACE2
Angiotensin (1-9)
ACE
Angiotensin (1-7)
Mas / (AT1-7R?)
Reporter
Viral Constructs: single vector
(+ Dox)
EF1a
rtTA IRES tTS
TRE
PLAP poly A
EF1a - elongation factor 1 alpha
rtTA - “Tet-ON”
IRES - internal ribosome entry site
tTS - tet-silencer
TRE - tetracycline responsive element
PLAP - placental alkaline phosphatase
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
Reporter
Viral Constructs: two vectors
EF1a
rtTA IRES tTS poly A
(+ Dox)
TRE
SEAP poly A
EF1a - elongation factor 1 alpha
rtTA - “Tet-ON”
IRES - internal ribosome entry site
tTS - tet-silencer
TRE - tetracycline responsive element
SEAP - secreted alkaline phosphatase
Two vectors in vitro
140
Luminescence (RLU)
130
No Doxycycline
(1 ug/uL) Doxycycline
120
110
100
90
80
(Detection Limit)
70
60
TRE-SEAP
EF1a-rtTA-IRES-tTS
and TRE-SEAP
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?
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?)
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
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