CARDIOVASCULAR DISEASES

RECOMBINANTDNA TECHNOLOGYSESSION-2016-17

GENE THERAPYFOR CARDIOVASCULARDISEASES

CARDIOVASCULAR DISEASESInvolve heart or blood vesselsLeading cause of death globallyIncludes 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.

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

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.

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.

GENE THERAPY

Gene therapy - Therapeutic delivery of nucleic acid polymers into a patients 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.

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.

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.

VECTORS FOR CV GENE THERAPYNon-Viral VectorsPlasmidUltrasound-targetted Microbubbles(UTM)Cationic LiposomesViral VectorsAdenoviral(Ad) VectorsAdenoassociated Viral(AAV) VectorsLentiviral Vectors

Plasmid TransfectionApplied 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.

FIG: STRUCTURE OF PLASMID pBR322

CATIONIC LIPOSOMESNegatively 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.

CATIONIC LIPOSOMES

ULTRASOUND-TARGETTED MICROBUBBLESIntrinsic low levels of toxicity and immunogenicityPotential for re-administration and organ-specific delivery of the genes of interest.Enhance delivery of microRNAs to cardiomyocytes without discernable toxicity.

FIG.: Engineering Virus into Vector.

ADENOVIRAL VECTORSAdenoviral (Ad) vectors are non-enveloped, non-integrating doublestranded (ds)DNA vectorsEnter 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 (12 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.

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.

ADENOVIRAL VECTOR

ADENO-ASSOCIATED VIRAL VECTORSAAV are single-stranded (ss)DNA vectors.Provoke much less inflammationMore than 100 serotypes of the wild-type AAVAAV1, AAV6, AAV8, and AAV9 have been identified as the most cardiotropic serotypesAAV9 was the most efficient serotype for cardiac gene delivery in mice.

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.

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.

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 v 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 v5 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.

LENTIVIRAL VECTORSLV 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.

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.

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).

ENHANCING UPTAKE OF VIRAL VECTORSAdjuvants 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.

REGULATION OF GENE EXPRESSIONExpression 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.

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.

TET-OFF AND TET-ON SYSTEM

BARRIERS OF GENE THERAPY FOR CARDIOVASCULAR DISEASESGene 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.

UTMD IN GENE THERAPYUltrasound 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.

UTMD FOR CVDsMicrobubbles 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.

ADVANTAGES OF UTMDMicrobubbles 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

MECHANISM OF UTMD

GENE THERAPY FOR HEART FAILUREHEART FAILURE-When theheartis unable to pump sufficiently to maintainblood flowto 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 -adrenergic receptor kinase (ARKct)Parvalbumin (PVALB).

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.

ATHEROSCLEROSISHardening and Narrowing of the arteries-silently and slowly blocks arteries, putting blood flow at risk.Atherosclerosis begins with damage to the endothelium. Its caused byhigh blood pressure,smoking, or highcholesterol. 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.

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.

ISCHAEMIAImpaired 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.

ISCHAEMIA

CONTD.Ad mediated transfer of VEGF has been demonstrated to improve the endothelial function

FIG.: Angiogenesis in the ischaemic myocardium

Angiogenesis in the ischaemic myocardiumThe angiogenic growth factors bind to specific receptors located on the endothelial cells (EC) of nearby preexisting blood vessels.Activation of EC by VEGF.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.As the vessel extends, the tissue is remoulded around the vessel and proliferating endothelial cells roll up to form a blood vessel tubeBlood vessel loops are formed from individual blood vessel tubes and these are stabilized by the formation of SMC.

THROMBOSISFormation 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 aggregationThe 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

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

DEEPVEIN THROMBOSIS

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 666679 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 dATPlevels using gene therapy

OVERVIEWOmecamtiv 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.

OVERVIEWdATP 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

GENE-DELIVERY SYSTEMWhen ratcardiomyocytes 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

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.

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-R2treated cells(triangles)showed significantly greater response to Ca2+at all frequencies. AV-GFPtreated cells(open circles); nontransduced cells(solid circles). *p< 0.05 compared with nontransduced; p< 0.05 compared with AV-GFPtreated.(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-R2treated neonatal rat ventricular myocytes (NRVMs).(D)Increased intracellular dATP in AV-R1+ AV-R2treated NRVMs. *p< 0.05 compared with AV-GFPtreated 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 666679

CONCLUSIONGene 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.

REFERENCESJurez JLR and Herzberg AZ. Gene Therapy in Cardiovascular Disease. Gene Therapy ApplicationsDishart KL, Work LM,Denby L, and Baker AH. Gene Therapy for Cardiovascular Disease. Journal of Biomedicine and Biotechnology.2003:2 (2003) 138148Chen ZY, Lin Y, Yang F, Jiang Land Ge SP. Gene therapy for cardiovascular disease mediated by ultrasound and microbubbles. Chen et al. Cardiovascular Ultrasound 2013, 11:11Rincon MY, Driessche TV, and Chuah MK. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation. Cardiovascular Research (2015) 108, 420 doi:10.1093/cvr/cvv205Wolfram JA, Donahue JK. Gene Therapy to Treat Cardiovascular Disease. Journal of the American Heart Association. DOI: 10.1161/JAHA.113.000119NET SOURCE- wikipedia.org1

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