The document discusses gene therapy for cardiovascular diseases, outlining various conditions including heart disease, atherosclerosis, and thrombosis. It explores gene therapy mechanisms, particularly focusing on different vectors like adenoviral and lentiviral vectors, as well as recent advancements such as ultrasound-targeted microbubble destruction (UTMD) to enhance gene delivery. Additionally, the challenges and potential solutions in overcoming barriers to effective gene therapy in treating heart failures and ischemia are examined.

1. RECOMBINANT
DNA TECHNOLOGY
BANASTHALI UNIVERSITY
SUBMITTED BY:
• AKANKSHA BISHT
• BTECH(BT)-VI SEM
• ROLL NO. 8334
SUBMITTED TO:
• DR. MOUSUMI DEBNATH
• ASSOCIATE PROFESSOR
• DEPT. OF BIOSCIENCE AND
BIOTECHNOLOGY
SESSION-2016-17

2. GENE THERAPY
FOR
CARDIOVASCULAR
DISEASES

3. CARDIOVASCULAR
DISEASES
•Involve heart or blood vessels
•Leading cause of death globally
•Includes coronary artery diseases (angina and
myocardial infarction),stroke, heart failure,
hypertensive heart disease, rheumatic heart
disease, cardiomyopathy, heart arrhythmia,
congenital heart disease, valvular heart
disease, carditis, aortic aneurysms, peripheral
artery disease, and venous thrombosis.

4. CVDs involving Blood Vessels
• Coronary artery disease (also known as
ischemic heart disease)
• Peripheral arterial disease – disease of blood
vessels that supply blood to the arms and legs
• Cerebrovascular disease – disease of blood
vessels that supply blood to the brain (includes
stroke)
• Renal artery stenosis
• Aortic aneurysm

5. CVDs Involving the Heart
• Cardiomyopathy – Diseases of cardiac muscle.
• Hypertensive heart disease – Diseases of the
heart secondary to high blood pressure or
hypertension.
• Heart failure - A clinical syndrome caused by the
inability of the heart to supply sufficient blood to
the tissues to meet their metabolic requirements.
• Pulmonary heart disease – A failure at the right
side of the heart with respiratory system
involvement.
• Cardiac dysrhythmias – Abnormalities of heart
rhythm.

6. CONTD.
•Inflammatory heart disease
Endocarditis – Inflammation of the inner layer of
the heart, the endocardium. The structures most
commonly involved are the heart valves.
Inflammatory cardiomegaly
Myocarditis – inflammation of the myocardium, the
muscular part of the heart.
• Valvular heart disease
• Congenital heart disease – Heart structure
malformations existing at birth.
• Rheumatic heart disease – Heart muscles and valves
damage due to rheumatic fever caused by Streptococcus
pyogenes a group A streptococcal infection.

9. GENE THERAPY
• Gene therapy - Therapeutic delivery of nucleic
acid polymers into a patient’s cells as a drug to
treat disease.
• The most common form uses DNA that encodes a
functional, therapeutic gene to replace a mutated
gene. The polymer molecule is packaged within a
"vector", which carries the molecule inside cells.
• DNA must be administered, reach the damaged
cells, enter the cell and express/disrupt a protein.

10. FIG: Gene therapy using an adenovirus vector
In some cases, the adenovirus will insert the new gene into a cell. If the treatment is
successful, the new gene will make a functional protein to treat a disease.

11. FIG: After the insertion of the therapeutic gene into a genetic vector, the gene is
transferred to the body with the in vivo or the ex vivo technique.

12. VECTORS FOR CV GENE THERAPY
Non-Viral Vectors
• Plasmid
• Ultrasound-targetted Microbubbles(UTM)
• Cationic Liposomes
 Viral Vectors
• Adenoviral(Ad) Vectors
• Adenoassociated Viral(AAV) Vectors
• Lentiviral Vectors

13. PLASMID TRANSFECTION
• Applied only in the case of direct needle
injection (puncture) in specific muscular
tissues.
• Inability to present a controlled and
satisfactory expression of the genes that they
carry in special target-tissues.
• May carry any volume of genetic material to
the target-cells without any restriction.

14. FIG: STRUCTURE OF PLASMID pBR322

15. CATIONIC LIPOSOMES
• Negatively charged DNA is contained within a positively
charged lipid vesicle. Plasmid DNA is released in the
cytoplasm, but only a small proportion of it enters the
nucleus.
• Liposomes as genetic vectors, carry on their surface
some substances attached, recognized by special
receptors of specific cell populations in the body.
• The selective introduction of liposomes in the target-
cells is thus achieved through endocytosis, following
the mediation of the respective cellular receptor.
Asialoglycoprotein, transferin etc. have been used as
such surface molecules.

16. CATIONIC LIPOSOMES

17. ULTRASOUND-TARGETTED
MICROBUBBLES
• Intrinsic low levels of toxicity and
immunogenicity
• Potential for re-administration and organ-
specific delivery of the genes of interest.
• Enhance delivery of microRNAs to
cardiomyocytes without discernable toxicity.

18. FIG.: Engineering Virus into Vector.

19. ADENOVIRAL VECTORS
• Adenoviral (Ad) vectors are non-enveloped, non-
integrating doublestranded (ds)DNA vectors
• Enter the cells predominantly via clathrin-mediated
endocytosis upon binding with coxsackie-adenovirus
receptor(CAR).
• In the heart, transgene expression after Ad vectors
transduction is robust but transient (1–2 weeks),24,25
which limits its applications in CVD for HF.
• Useful system for short-term pro-angiogenic therapies
in ischaemic heart disease,26 peripheral arterial
occlusive disease, and limb ischaemia.

20. CONTD.
• DISADVANTAGE: Ability to induce Inflammation.
• The early-generation Ad vectors contain residual
adenoviral genes in the vector backbone that
trigger T-cell mediated immune responses that
eliminate the gene-modified cells.
• Recombinant vectors derived from the serotype 5
adenovirus (Ad5) are used.
• The CAR is the primary cell surface receptor for
Ad5.

21. ADENOVIRAL VECTOR

22. ADENO-ASSOCIATED VIRAL VECTORS
• AAV are single-stranded (ss)DNA vectors.
• Provoke much less inflammation
• More than 100 serotypes of the wild-type AAV
• AAV1, AAV6, AAV8, and AAV9 have been
identified as the most cardiotropic serotypes
• AAV9 was the most efficient serotype for
cardiac gene delivery in mice.

23. CONTD.
• AAV9 transduction is not restricted to the myocardium,
since other tissues, including liver and skeletal muscle
can also be transduced.
• Using an AAV cap gene library produced by DNA
shuffling of different AAV serotype capsid genes, Yang
et al. obtained a myocardium-tropic AAV strain,
AAVM41,that exhibited enhanced transduction to
cardiac muscle and diminished tropism to the liver
after systemic administration.
• Variants AAV9.45 and AAV9.61 that displayed a 10- to
25-fold lower gene transfer efficiency in liver, while
transducing the myocardium as efficiently as AAV9.

24. CONTD.
• MAJOR DRAWBACK: Limited packaging capacity of
the vector particles (i.e. 4.7 kb), which constrains the
size of the transgene expression cassette that can be
used.
• Dual vector strategies have been developed to
overcome the packaging constraints.

26. FIG.: (a) Adenoviral (Ad) attachment and internalisation is mediated through the knob protein of the
fiber binding to CAR, followed by interaction of the penton base at the base of the fiber shaft with αv
integrins on the cell surface. Following internalisation, the virus is localised within cellular endosomes
which upon acidification allows the virions to escape and traffic to the nucleus. Admediated infection is
therefore, dependent on levels of CAR with hepatocytes being highly permissive as shown with
reasonable levels of transduction in endothelial cells (EC). (b) AAV2 binds to the primary receptor
heparin sulfate proteoglycan (HSPG) on the cell surface and internalization is assisted by the secondary
receptors αvβ5 integrins and fibroblast growth factor receptor 1. Transduction of vascular cell, in
particular EC, is very poor compared with permissive cell types such as HeLa. Transduction of both cell
types with rAAV2-eGFP clearly shows the difference in transduction efficiency.

27. LENTIVIRAL VECTORS
• LV are derived from the HIV type1 (HIV-1).
• LV are enveloped single-stranded (ss)RNA
vectors.
• Have the ability to stably integrate their
genome as cDNA into the chromosomes of
both dividing and non-dividing target cells.
• Therefore,well suited to achieve long-term
expression of the therapeutic gene.

28. CONTD.
• Since LV could also transduce endothelial cells or
endothelial progenitors, these properties have
potential implications for the treatment of
peripheral vascular diseases by LV gene therapy.
• Since they can integrate randomly into the target
cell genome, with a preference for genes, their
use carries an intrinsic risk of triggering
Insertional Oncogenesis.
• This risk can be reduced by optimizing the vector
design and depends on the target cell type.

30. FIG.: Strategies to increase cardiac-specific gene transfer using AAV vectors using naturally occurring
AAV serotypes, AAV capsid engineering, or directed molecular evolution and in vivo selection of
cardiotropic AAV variants (see text for details). The relative cardiac transduction efficiency of the
naturally occurring AAV serotypes (AAV2, AAV1, AAV6, AAV8, and AAV9).

31. ENHANCING UPTAKE OF VIRAL VECTORS
• Adjuvants enhance vector uptake into the
myocardium.
• Sasano et al.90 reported an increase of up to
80% in cardiac transduction efficiencies in a
porcine model by using a combination of
VEGF, adenosine, calcium, and nitroglycerin
(NTG) infusion prior to the viral vector
administration.

32. REGULATIONOF GENE EXPRESSION
• Expression levels of an introduced gene depend mostly
on the transduction efficiency of the vector and on the
strength of the transcriptional regulatory elements.
• Strong and ubiquitously active viral promoters such as
human cytomegalovirus (CMV) used to drive transgene
expression.
• The tetracycline-controllable expression system
enables tight on/ off regulation, high inducibility, fast
response times, no pleitropic effect owing to the use of
the tetracycline operon derived from bacteria, and a
well-characterized inducer, namely, tetracycline or
doxycycline.

33. CONTD.
• Two types of system have been used to regulate
transgene expression: tet-off and tet-on.
• tet-off system: tetracycline-responsive
transcriptional activator (tTA) induces the
transcription of a gene containing the tet-
responsive element, and transcription is turned
off in the presence of tetracycline.
• tet-on system: the reverse tetracycline-
responsive transcriptional activator (rtTA), binds
to the tet-responsive element and turns on the
transcription in the presence of tetracycline.

34. TET-OFF AND TET-ON SYSTEM

35. BARRIERS OF GENE THERAPY FOR
CARDIOVASCULAR DISEASES
• Gene vectors need to pass through the endothelial
barriers in capillary walls when systemically injected.
• Plasmid faces a threat of being degrade rapidly by the
immune system or DNAse in serum before transfection.
• Viral gene vectors need to avoid the immunoreaction
in circulation and transduction of non-target organs,
mainly liver and spleen.
• Plasmid needs to avoid being entrapped into lysosome
or the endosome, where it will be degraded.
• Gene vector has to penetrate the nuclear membrane to
achieve the goal of gene therapy.

36. UTMD IN GENE THERAPY
• Ultrasound Targetted Microbubble Destruction (UTMD) could
enhance transfection efficiency of naked plasmid DNA by several
orders of magnitude.
• UTMD is based on the specific response of the microbubbles upon
exposure to ultrasound, namely sonoporation.
• Microbubbles(MBs) of UTMD, which may consist of lipids, albumin,
saccharide, biocompatible polymers and other materials are
traditionally used as ultrasound contrast agents due to their
physical property of reflecting ultrasound.
• Microbubble as cavitation nucleus could expand and contract under
the effect of ultrasound, and even be disrupted when the acoustic
pressure reaches a much higher level.
• The gene therapy vector incorporated with microbubbles can be
released with high local concentrations at the site of interest.

37. UTMD FOR CVDs
• Microbubbles carrying therapeutic gene are destroyed at the site of the
target tissue, resulting in sonoporation and delivery of the drug directly to
the target cell.
• The process of sonoporation
induced by US application leads
to transientl holes in cell
membrane and capillary, which
facilitates the uptake of
therapeutic gene.

38. ADVANTAGES OF UTMD
• Microbubbles offer the strength of site specific
release through ultrasound irradiation, thus
improving viral vector specificity.
• Production of microjet by UTMD can enhance the
penetrability of plasma membrane and capillary,
thus overcoming the endothelial barrier.
• Microbubbles can simultaneously impose
restriction on the immune response to the
viruses thus allowing intravascular administration
and repetitive injections

39. MECHANISM OF UTMD

40. GENE THERAPY FOR HEART FAILURE
 HEART FAILURE-When the heart is unable to pump
sufficiently to maintain blood flow to meet the body's
needs.
 Molecular targets for gene therapy:
• Sarcoendoplasmic reticulum calcium-ATPase 2a (SERCA2a)
• Stromal-derived factor-1 (SDF-1)
• Adenylyl cyclase 6 (ADCY6)
• S100 calcium-binding protein A1 (S100A1)
• A c-terminal fragment of the β-adrenergic receptor kinase
(βARKct)
• Parvalbumin (PVALB).

41. CONTD.
• One of the key proteins defective in HF is SERCA2a.
• SERCA2a expression and function are decreased in
heart failure. This decrease reduces calcium transient
that is characteristic of systolic heart failure.
• The Calcium Upregulation by Percutaneous
administration of gene therapy In cardiac Disease
(CUPID) trial looked at the safety and efficacy of
SERCA2a gene therapy in HF.
• In this, infusion of recombinant AAV-1 encoding
SERCA2a is done.

43. ATHEROSCLEROSIS
• Hardening and Narrowing of the arteries-silently and slowly blocks
arteries, putting blood flow at risk.
• Atherosclerosis begins with damage to the endothelium. It’s
caused by high blood pressure,smoking, or high cholesterol. That
damage leads to the formation of plaque.
• A reduction in the level of atherogenic apolipoprotein (apo) B100
is possible after gene transfer of the apoB mRNA editing enzyme,
whilst lipoprotein A could be lowered with synthesis inhibiting
ribozymes.
• Apolipoprotein AI (apoAI) and lecithin-cholesterol acyltransferase
(LCAT) are important factors in the removal of excess cholesterol
and the subsequent reduction in the incidence of atherosclerotic
lesions.

44. CONTD.
• Through in vitro Bicistronic Expression of these
two genes from AAV plasmid vectors, it was
shown that increased synthesis of apoAI and
LCAT could play a role in reducing atherosclerotic
risk.

45. ISCHAEMIA
Impaired blood supply resulting from narrowed
or blocked arteries, which subsequently starve
tissues of the necessary nutrients and oxygen.
Two main therapeutic genes under investigation
are:
• Angiogenic Growth Factors (VEGF)
• Fibroblast Growth Factor (FGF).
VEGF is a heparin binding glycoprotein, which is
a principal angiogenic factor for endothelial cells.

46. ISCHAEMIA

47. CONTD.
• Ad mediated transfer of VEGF has been
demonstrated to improve the endothelial
function
FIG.: Angiogenesis in the
ischaemic myocardium

48. ANGIOGENESIS IN THE ISCHAEMIC
MYOCARDIUM
1. The angiogenic growth factors bind to specific receptors located
on the endothelial cells (EC) of nearby preexisting blood vessels.
2. Activation of EC by VEGF.
3. Synthesis of new enzymes is triggered. These enzymes dissolve
tiny holes in the sheath-like covering surrounding all existing
blood vessels. The endothelial cells proliferate and migrate out
through the dissolved holes of the existing vessel.
4. As the vessel extends, the tissue is remoulded around the vessel
and proliferating endothelial cells roll up to form a blood vessel
tube
5. Blood vessel loops are formed from individual blood vessel tubes
and these are stabilized by the formation of SMC.

49. THROMBOSIS
• Formation of a blood clot inside a blood vessel,
obstructing the flow of blood through the circulatory
system.
• Reduction of antithrombotic activity leading to clot
formation.
• Prostacylin (PGI2), nitric oxide (NO) and thrombin
inhibitors all act through the inhibition of platelet
adhesion and aggregation
• The anti-thrombotic treatment, tissue plasminogen
activator (tPA), which has anticoagulant properties and
is used to lyse existing clots, may be a useful
therapeutic gene for antithrombotic therapy

50. CONTD.
• Other anticoagulant gene products include hirudin,
thrombomodulin, antistasin and, tissue factor
pathway inhibitor (TFPI).
• Hirudin - Most potent inhibitor of thrombin, the
enzyme responsible for fibrinogen cleavage,platelet
activation, and SMC proliferation.
• Cyclo-oxygenase-1 (COX-1), the rate limiting enzyme in
the synthesis of PGI2, was overexpressed by local
delivery of Ad to porcine carotid arteries immediately
postangioplasty. This was shown to increase the levels
of PGI2 and, in turn, inhibit thrombosis in injured
vessels

51. DEEP–VEIN THROMBOSIS

52. RECENT STUDY
Thomson KS, Odom GL,Murry CE, Mahairas GE, Harami FM, Teichman SL,
Chen X, Hauschka SD, Chamberlain JS, Regneir M.Translation of Cardiac
Myosin Activation with 2-Deoxy-ATP to treat Heart Failure via an
Experimental Ribonucleotide Reductase based Gene Therapy . JACC: Basic to
Translational Science. Volume 1, Issue 7, December 2016, Pages 666–679
ABSTRACT
CHRONIC HEART FAILURE: Growing cause of morbidity, mortality,
hospitalizations.
The authors discovered that small amounts of 2-deoxy-ATP (dATP) activate
cardiac myosin leading to enhanced contractility in normal and failing heart
muscle.
Cardiac myosin activation triggers faster myosin cross-bridge cycling with
greater force generation during each contraction.
The authors studied the translational medicine effort to increase dATP levels
using gene therapy

53. OVERVIEW
• Omecamtiv mecarbil is the only cardiac myosin-
activating drug currently in development.
• Development of BB-R12 (AAV6 viral vector with a
cardiac-specific promoter cTnT455 to overexpress
R1R2 [ribonucleotide reductase, containing R1 (Rrm1)
and R2 (Rrm2) subunits] in the heart).
• This targets myocardial contractility directly by
increasing production of 2-deoxy-ATP (dATP) in
cardiomyocytes.
• R1 subunit contains the catalytic site and 2 allosteric
sites that can bind dATP, whereas the smaller R2
subunit contains the free radical generator.

54. OVERVIEW
• dATP concentration can be increased by overexpression of
R1R2, the rate-limiting enzyme in its production, using gene
therapy.
• BB-R12 is a designed multicomponent gene therapy agent
consisting of a recombinant serotype-6 adeno-associated
viral vector (AAV6) carrying a genome containing a human
cardiac troponin T regulatory cassette (hcTnT455) linked to
a transgene encoding human sequences of both the large
(R1) and small (R2) subunits of R1R2 to overexpress the
enzyme in myocardium.
• BB-R12 creates a drug production and delivery system
within the heart

55. GENE-DELIVERY SYSTEM
• When rat cardiomyocytes were transduced with AV-R1 +
AV-R2, intracellular dATP content, magnitude, and rate of
contraction and relaxation all increased, without affecting
Ca2+ transient properties. These results suggest that the
increased contractility seen with R1R2 overexpression is
due to increased myofilament responsiveness to Ca2+.
• This study provided the first proof of principle that dATP
levels could be increased in cardiomyocytes and that small
elevations of dATP levels enhanced contractility.
• When the AV-R1 + AV-R2 system was used analogously to
transduce human cells (human embryonic stem cell-
derived cardiomyocytes [hESC-CMs]), contractility was
again significantly increased

56. CONTD.
• Transfer of small molecules such as ATP and dATP between
cells is facilitated by gap junctions.
• dATP could diffuse through gap junctions between
physically coupled cardiomyocytes to enhance contractility
of neighboring cells that were not overexpressing R1R2.
• To test this, the transfer of fluorescein-labeled dATP via gap
junctions between AV-R1 + AV-R2 transduced and
nontransduced (WT) cardiomyocytes (rat and human) and
the resulting effects on contractility were measured.
• Rapid transfer of dATP between coupled cells was
demonstrated, and this effect was blocked with a gap
junction inhibitor.

57. • FIG.: Effects of AV-R1 + AV-R2 Treatment on ARCs
(A) Contractile response of adult rat cardiomyocytes (ARCs) at different
stimulation frequencies. AV-R1 + AV-R2–treated cells (triangles) showed
significantly greater response to Ca2+ at all frequencies. AV-GFP–treated
cells (open circles); nontransduced cells (solid circles). *p < 0.05 compared
with nontransduced; †p < 0.05 compared with AV-GFP–treated. (B to D) R1R2
protein expression in ARCs after AV-R1 + AV-R2 treatment. Increased
R1 (B) and R2 (C) protein expression in AV-R1 + AV-R2–treated neonatal rat
ventricular myocytes (NRVMs). (D) Increased intracellular dATP in AV-R1 + AV-
R2–treated NRVMs. *p < 0.05 compared with AV-GFP–treated NRVMs. GFP =
green fluorescent protein
REF: Thomson KS, Odom GL,Murry CE, Mahairas GE, Harami FM, Teichman SL, Chen X, Hauschka SD, Chamberlain JS, Regneir M.Translation
of Cardiac Myosin Activation with 2-Deoxy-ATP to treat Heart Failure via an Experimental Ribonucleotide Reductase based Gene Therapy .
JACC: Basic to Translational Science. Volume 1, Issue 7, December 2016, Pages 666–679

58. CONCLUSION
• Gene therapy is emerging as a suitable alternative, with substantial
progress in preclinical models of CVD.
• However, the ability to obtain sustained expression of the gene of
interest may not always be warranted and sometimes transient
expression may be preferred based on safety considerations.
• The physiological and structural differences between animal models
and humans and the development of immune response against the
transgene products, the gene-modified cells, or the vectors
themselves pose important challenges for clinical translation.
• In order for gene therapy to become a reality in the cardiovascular
clinic, effective therapeutic genes and suitable vectors must be
identified and developed.
• The hope is that the majority of patients who suffer from less
severe heart disease may ultimately benefit from the advances in
gene therapy.

59. REFERENCES
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10.1161/JAHA.113.000119
• NET SOURCE- wikipedia.org1

60. THANK YOU