VEGFA – A POTENTIAL GENE THERAPY TARGET FOR CARDIAC ISCHEMIA

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume:02/Issue:09/September-2020

Impact Factor- 5.354

www.irjmets.com

VEGFA – A POTENTIAL GENE THERAPY TARGET FOR CARDIAC ISCHEMIA Aindrilla Roy*1, Arsalan Hussain*2, Krishnendu Sinha*3, Mahima Praharaju*4, Saba Khatun*5, Ritik Raj*6 *1M.Tech,

Department of Bioprocess Engineering, IIT Roorkee,Uttarakhand, India.

*2M.Sc

Semester 3, Department of Life Sciences, Presidency University, Kolkata, West Bengal, India.

*3M.Sc

Semester 3, Department of Life Sciences, Presidency University, Kolkata, West Bengal, India.

*4B.Sc *5M.Sc,

,Department of Life Sciences, Acharya Nagarjuna University, Andhra Pradesh, India.

Department of Microbiology, Assam Don Bosco University, Kamarkuchi, Assam, India.

*6M.Pharm,

Department of Pharmaceutical Biotechnology, Bharati Vidyapeeth University, Pune., Maharashtra , India.

ABSTRACT Gene therapy is the insertion of genes into an individual’s cells or tissues to treat diseases where deleterious mutant alleles are replaced with functional ones. Amongst the diseases of the heart, cardiac ischemia has the highest number of reported cases worldwide. Myocardial ischemia takes place due to an inadequate supply of blood flow to the heart muscles or myocardium often leading to a heart attack.

Amidst the standard treatments available, gene therapy has proved to be the most promising one with a high success rate. This article discusses the restorative potential of VEGFA gene therapy for cardiac ischemia and its upcoming prospectives; plasmid vectors showed lesser side effects with gene electrotransfer or electroporation being the gene delivery method with a soaring productive outcome.

KEYWORDS : Gene therapy, vectors ,cardiac ischemia, gene delivery, VEGFA, vector.

I.

INTRODUCTION

Gene therapy refers to the treatment of genetic diseases by transferring normal functional copies of the gene into the cell that acts as a potential cure for any genetic diseases. Many acquired or inherited gene defects can be subjected to treatment by therapeutic insertion of genetic material within the target cell using vectors (viral or non-viral or adenoviral), or simply by using tools of genome editing like CRISPR, TALENs, or ZFNs. The first approved gene therapy in the United States took place on September 14, 1990, at the NIH. A four-year girl named Ashanti DeSilva was treated for a genetic defect that left her with an Immune System deficiency (SCID) at the National Institute of Health (NIH). The effects were only temporary. Having said that, it has set a milestone in the history of gene therapy, and since then over 2600 clinical trials took place in patients with various genetic defects.

In general, there are two types of gene therapy – germline gene therapy and somatic gene therapy. In the former one, germ cells, i.e., sperms or eggs are modified by the introduction of functional genes, which are integrated with their genomes. Targeting of germ cells makes the therapy heritable. While in the latter, the therapeutic genes are transferred into the somatic cells of the patient and thus, the effects of the foreign gene are restricted to an individual patient only.

Gene therapy for Cardiovascular Diseases Cardiovascular diseases have always been a global health problem. Several conventional methods are made available to treat a few common CVDs, whereas some remained with unmet clinical needs. Gene therapy has a high capacity to modify the genes that reduce cardiac problems and has proven its effectiveness in tissues, both in vivo and in vitro. The fact that the long term results obtained from the application in tissues with negative side effects are an added advantage. To improve the main objective, enhancement of the viral vector responsible for the delivery of genetic material is required. The most

www.irjmets.com

@International Research Journal of Modernization in Engineering, Technology and Science

[208]

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume:02/Issue:09/September-2020

Impact Factor- 5.354

www.irjmets.com

studied targets are the blood vessels since they are easy to access. A recent study shows that the CRISPRcas gene is used in both cardiac and vascular areas.

Figure 1. Delivery vehicles and routes for cardiac gene therapy. A Schematic representation of the main delivery strategies for cardiac gene therapy (injection of naked plasmid DNA or gene transduction using adenoviral or adeno-associated virus [AAV]-based vectors).

The approximate size of the delivery vehicle is indicated. B, Main delivery routes to reach the heart. These include injection into the coronary artery as during standard percutaneous coronary intervention or retrograde into the coronary sinus, on the left side panel; or intramyocardial, on the right-side panel, through either direct injection after mini-thoracotomy or during bypass surgery or after percutaneous catheterization to reach the left ventricle, followed by

Table-1: In-detail gene therapy for cardiovascular diseases.

II.

METHODOLOGY

Gene therapy for Myocardial ischemia (in-vivo): Myocardial ischemia occurs when blood flow to the heart is decreased due to partial or complete blockage of coronary arteries preventing the heart from receiving enough oxygen. Gene therapy for cardiac ischemia involves gene electrotransfer, an in-vivo method to deliver the plasmid encoding VEGF-A www.irjmets.com

@International Research Journal of Modernization in Engineering, Technology and Science

[209]

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume:02/Issue:09/September-2020

Impact Factor- 5.354

www.irjmets.com

to the ischemic heart. In this in-vivo technique, the cloned genes are transferred directly into the patient’s tissues, whose cells cannot be cultured in-vitro in sufficient numbers and/or where re-implantation of cultured cells is not efficient. Though the primary methods of gene delivery for cardiac ischemia are viralmediated and plasmid-based transfers, some vectors like adenoviral and adeno-associated (AVV) vectors are also frequently used with major side effects like immunogenicity and toxicity. Plasmid-mediated delivery comes with a few side effects like low immunogenicity and toxicity at a reduced cost but with lower efficiency.

Gene therapy has been more efficient in the successful delivery of the transgene into the defective cell, owing to the vectors that act as a vehicle or an agent in the process. Vectors have 2 main categories which are further divided- viral and non-viral. Viral vectors are very broad and include both long and short term expression, representing both RNA&DNA viruses either single-stranded or double-stranded genomes.

The major viral vectors are adenoviral and adeno-associated vectors (AVV). Adenoviral vectors come with a wide range of host cells and strong immunogenicity, while AAV has a moderate immune response.

So far the usage of non-viral vectors has been shadowed with the viral-vectors always being under the spotlight. They are far less immunogenic than viral-vectors. With that being said, the development of nonviral DNA vectors has progressed steadily, remarkably in the case of plasmids. They are now allowed to fill in where viral and other non-viral vectors may not be the best options. Plasmids are easier and cheaper to produce, ship, and store with much longer shelf-life. The modular nature of plasmids also allows uncomplicated molecular cloning, making them easy to manipulate and design for therapeutic use.

Neoteric studies indicate, the smaller the plasmid size, the greater the transfection efficiency. Initial attempts for the optimization of plasmid DNA vectors for gene therapy involved the removal of antibioticresistant genes, thus enhancing their clinical potential.

This led the antibiotic-free systems to utilize a different mechanism for selection. One of the first systems was the operator and repressor titration (ORT). These plasmids (pORT) contain one or more operator sequences that are used to the filtrate, through competition and repressor proteins (Lac repressor) that bind to an endogenous operator sequence for the upstream of a chromosomally encoded and essential gene of bacteria. Here, in pORT, the gene is dap4. Since there are very few bacterial sequences encoded on the plasmid it requires no plasmid gene expression. It promotes stability and if the need is for production, can be used in any microorganism that will be able to get through the plasmid. Other systems include the conditional origin of replication (pCOR) and free antibiotic resistance (pEAR). The usage of plasmid vectors comes with some limitations as well. As they are non-replicating episomes, transgene expression is transient and diluted by cell division. Sometimes, residual antibiotics from vector production can also trigger an immune response in patients, Due to these drawbacks, extensive modifications are made in the plasmids to satisfy regulatory requirements for clinical use in humans.

Figure-2: Clinical trials for therapeutic angiogenesis. The figure summarizes the main clinical trials for therapeutic angiogenesis, grouped according to the delivery method used (naked plasmid DNA, top or adenoviral vectors, bottom), along with the indication of the therapeutic gene and the trial name. VEGF-A indicates vascular endothelial growth factor-A. www.irjmets.com

@International Research Journal of Modernization in Engineering, Technology and Science

[210]

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume:02/Issue:09/September-2020

III.

Impact Factor- 5.354

www.irjmets.com

ANALYSIS

Genome editing tools for Myocardial Ischemia: Clustered regularly interspaced short palindromic repeats-(CRISPR-) associated 9 (CAS9) is an on-off injection that has the potential to reduce the risk of cardiovascular disorders is now a prospect all thanks to the advancement in gene editing. The CRISPR/CAS9 genome editing system are now been extensively used to maximize the understanding of many cardiovascular diseases and are proven to be effective in creating gene knockouts or knock-in human cells. Vascular endothelial growth factor receptors(VEGFR)2 which promotes cardiac Angiogenesis is associated with many cardiovascular diseases that are being depleted by a system of adeno- associated virus(AAV)- mediated CRISPR/CAS9 from streptococcus pyogenes (SpCas9) in vascular endothelial cells. This work established a strong foundation for genome editing as a strategy to treat heart disease. Nowadays they have been used to evaluate the pathogenicity of Titin mutations in Dilated Cardiomyopathy.

Today the therapeutic potential of gene electrotransfer as a medium for plasmid encoding Vascular endothelial growth factor A delivery to Ischemia Myocardium in a porcine model is widely studied. A study was conducted to determine the safety of gene electrotransfer by injecting the VEGF-A encoding plasmid at four sites of surgically induced Myocardial Ischemic heart in a porcine. The state was monitored for seven days along with a control with no electrotransfer. In the end, it was observed that there was a significant reduction of infarct area, and left ventricle contractility got improved in electrotransfer of the plasmid group concerning control. It was concluded from the experiment that genome editing through plasmid is the safest way for myocardial repair and regeneration.

IV.

APPLICATION : HOW IS THE GENE THERAPY BROUGHT TO USE TO TREAT THE ISCHEMIC HEART CONDITION?

As already stated in the paper before, ischemia is a major contributor to fatalities observed due to genetic mal-functioning of the cardiovascular genes like VEGF. We already know, gene therapy is a probable therapeutic approach to treat the disorder. But knowing about the transgene to be inserted and the vector delivery system is not enough to carry out the procedure successfully. An appropriate application route has to be chosen for the successful incorporation of the transgene into the targeted cell. Broadly the routes can be divided into two; systemic application and regional application. The regional application can be further sub-divided into intra-myocardial injection, the anterograde, and retrograde infusion [1].

Catheter-based intra-cardiac injection: catheter used t directly inject into the endocardium over the left ventricle.

Systemic intravenous injection: feasible only if the vector is tropistic towards cardiac tissues or when large scale production is possible.

Anterograde injection into the coronary artery: performed with the help of a trans-luminal catheter; or with partial occlusion of the artery.

Very much useful for the gene therapy approach, the only threat is being posed by the plaques formed due to ischemia.

Retrograde (local) intravenous injection: mediated using SSR catheter avoiding contamination. Gained appreciation for homogenous delivery of vectors and hence increasing their efficacy.

Open chest intra-cardiac injection: requires surgical interventions (e.g. bypass surgery).

Figure-3: Demonstration of detailed application of route www.irjmets.com

@International Research Journal of Modernization in Engineering, Technology and Science

[211]

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume:02/Issue:09/September-2020

Impact Factor- 5.354

www.irjmets.com

The gene that plays a major role in the treatment against ischemia is VEGF; it induces angiogenesis. There are 5 isoforms of VEGF expressed by the human genome. Amongst them, VEGF A and VEGF B maintain signaling through VEGF RECEPTOR 1 and VEGF RECEPTOR 2 to maintain blood vessel physiology and its constant development [2]. VEGF-165 heparin treated (the most important isoform of VEGF A) has been proved to induce angiogenesis in ischemic cardiac conditions [3]. In an experiment carried out in rat myocardial ischemic model by Xiaojin Hao et al., 2007, it was experimentally proven that adenovirus containing the transgene for VEGF-165 heparin treated was capable of enhancing VEGF A expression. The transfected plasmid was proven to increase the capillary density by 17-18 %. The left ventricular chamber, the one most adversely affected area in case of ischemia, had shown to improve the rate of functioning with the introduction of VEGF-165 heparin treated through the adenovirus delivery vehicle [3]. In a different set of experiments by David A Bull et al., 2003, a different delivery vehicle was used carrying the desired gene of interest (VEGF-165) in an ischemic rabbit model [4]. In the experiment, a lipopolymeric gene delivery system was evolved (Teplex DNA). This delivery system was proven to increase the efficacy of the transgene upon direct myocardial injection. The experimental data revealed that around 42% of the development was observed in ejection fraction after the delivery of the gene.

Through this experiment, it was shown that the VEGF activity was restored completely and was not shortlived as shown by the previous experiments. This restoration of VEGF activity ultimately resulted in the normal functioning of the left ventricular chamber. The works of Rene A Tio et al., 1999, have highlighted that using naked VEGF DNA along with VEGF proteins not only induce neovascularization but also enhance the collateral flow of blood in the left ventricular chamber [5].

In recent experimental work, magnetic nanoparticles were used to deliver the adenovirus plasmid containing the VEGF-165 transgene. The magnetic field of the nanoparticles guided them to the specific site and enhanced cardiac regeneration. The number of apoptotic cells declined drastically (65%) and the re-vascularization was induced to increase expression of VEGF-165 genes. When the western blotting was done taking samples from the left ventricular chamber, there was a 20% increase in the levels of the VEGF protein from the initial condition before the therapy started [6]. Similar studies were made using animal models for VEGF-B, VEGF-C, and VEGF-D. But successful clinical trials were possible only with VEGF-165, VEGF-121and VEGF-C. Therapeutic agent

Trial Name

Phase

Result

Adenovirus-mediated VEGF-165 is administered by intracoronary injection.

Kuopio Angiogenesis Trial (KAT)

2/3

Coronary perfusion was found to improve.

A naked plasmid containing VEGF165 was administered by intramyocardial injection.

EUROINJECT trial

2/3

Left ventricular function and wall motion was found to improve. But perfusion defects persisted. [8]

A naked plasmid containing VEGF165

NORTHERN TRIAL

2/3

Revascularization was observed but no perfusion improvement was seen.

[9]

Adenovirus-mediated VEGF-121 by injecting via myocardial followed by a small thoracotomy

REVASC trial

2

Perfusion and anginal symptoms were seen to improve. [10]

A naked plasmid containing VEGF 2 (VEGF C)

AGENT trial

½

Anginal symptoms improved and protection was observed for as long as 2 years. [11,12]

[7]

Table-2: Clinical trials in gene therapy of CVD’s www.irjmets.com

@International Research Journal of Modernization in Engineering, Technology and Science

[212]

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume:02/Issue:09/September-2020

V.

Impact Factor- 5.354

www.irjmets.com

CONCLUSION

The future of gene therapy in the treatment of cardiovascular diseases: Gene therapy is still an emerging branch of alternative therapeutic options and the vast amount of preclinical research trials reveals the enormous interest of researchers and clinicians in developing gene therapies as a potential cure for ischemic heart disease. Several upgrades in gene therapy technology have improved its efficacy and led it to gain success in animal models of ischemic heart disorders.

One such advancement is the use of nanoparticles as a vehicle for delivering the gene of interest to the target site which will soon replace the traditional virus-mediated gene transfer because the latter have safety issues associated with it which hinders its application from moving from animal models to clinical trials. Today with the knowledge of cell biology and the development of technologies, nanoparticles would be a desirable approach for future exploration.

Another possible avenue for cardiac gene therapy is the treatment of Arrhythmia a disease in which the electrical impulses of the heart do not work properly. In 2014 a study led by Cedars-Sinai Medical Center's cardiologist Eduardo Marban was able to successfully introduce a viral vector that carried specific porcine genes to stimulate the creation of a new pace making genes in pigs thereby creating a successful biological pacemaker. This study has not moved to human trials to date but shows a promising future-forward.

The current trials of gene therapy do not show any major safety concerns. This should encourage the conduction of more clinical trials to test the therapeutic effects of gene therapy in cardiovascular diseases.

ACKNOWLEDGEMENTS We gratefully thank our instructor Dr. Simranjit Singh for his insightful support and valuable guidance.

We also like to thank the team of Edufabrica and IIT Kharagpur for providing us with such a platform to expand our knowledge and gain lifelong experience.

VI.

REFERENCES

[1]

Bull, David A., et al. "Effect of Terplex/VEGF-165 gene therapy on left ventricular function and structure following myocardial infarction: VEGF gene therapy for myocardial infarction." Journal of controlled release 93.2 (2003): 175-181.

[2] Bulysheva, Anna A., et al. "Vascular endothelial growth factor-A gene electrotransfer promotes angiogenesis in a porcine model of cardiac ischemia." Gene therapy 23.8 (2016): 649-656.

[3] Celletti, Francesca L., et al. "Vascular endothelial growth factor enhances atherosclerotic plaque progression." Nature medicine 7.4 (2001): 425-429.

[4] Dishart, Kate L., et al. "Gene therapy for cardiovascular disease." Journal of Biomedicine and Biotechnology 2003.2 (2003): 138.

[5] Farraha, Melad, et al. "Gene therapy approaches to biological pacemakers." Journal of cardiovascular development and disease 5.4 (2018): 50.

[6] Gowdak, Luis Henrique W., et al. "Adenovirus-mediated VEGF121 gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of ishemia." Circulation 102.5 (2000): 565-571.

[7] Greenberg, Barry, et al. "Calcium upregulation by percutaneous administration of gene therapy in patients with cardiac disease (CUPID 2): a randomised, multinational, double-blind, placebocontrolled, phase 2b trial." The Lancet 387.10024 (2016): 1178-1186.

[8] Halonen, Paavo J., et al. "Current gene therapy trials for vascular diseases." Expert opinion on biological therapy 14.3 (2014): 327-336.

[9] Hao, Xiaojin, et al. "Myocardial angiogenesis after plasmid or adenoviral VEGF-A165 gene transfer in rat myocardial infarction model." Cardiovascular research 73.3 (2007): 481-487.

[10] Harris, J. D., et al. "Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E." Gene therapy 9.1 (2002): 21-29. www.irjmets.com

@International Research Journal of Modernization in Engineering, Technology and Science

[213]

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume:02/Issue:09/September-2020

Impact Factor- 5.354

www.irjmets.com

[11] Hedman, Marja, et al. "Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT)." Circulation 107.21 (2003): 2677-2683.

[12] Hulot, Jean-Sebastien, Kiyotake Ishikawa, and Roger J. Hajjar. "Gene therapy for the treatment of heart failure: promise postponed." European heart journal 37.21 (2016): 1651-1658.

[13] Ishikawa, Kiyotake, et al. "Cardiac I-1c overexpression with reengineered AAV improves cardiac function in swine ischemic heart failure." Molecular Therapy 22.12 (2014): 2038-2045.

[14] Jaffe, H. A., et al. "Adenovirus–mediated in vivo gene transfer and expression in normal rat liver." Nature genetics 1.5 (1992): 372-378.

[15] Juan, Shu-Hui, et al. "Adenovirus-mediated heme oxygenase-1 gene transfer inhibits the development of atherosclerosis in apolipoprotein E–deficient mice." Circulation 104.13 (2001): 1519-1525.

[16] Kastrup, Jens, et al. "Direct intramyocardial plasmid vascular endothelial growth factor-A165gene therapy in patients with stable severe angina pectoris: A randomized double-blind placebocontrolled study: The Euroinject One trial." Journal of the American College of Cardiology 45.7 (2005): 982-988.

[17] Katz, Michael G., et al. "Gene therapy in cardiac surgery: Clinical trials, challenges, and perspectives." The Annals of thoracic surgery 101.6 (2016): 2407-2416.

[18] Kim, Jun Sung, et al. "Intramyocardial transfer of hepatocyte growth factor as an adjunct to CABG: phase I clinical study." Gene Therapy 20.7 (2013): 717-722.

[19] Koeberl, Dwight D., et al. "Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors." Proceedings of the National Academy of Sciences 94.4 (1997): 1426-1431.

[20] Lavu, Madhav, Susheel Gundewar, and David J. Lefer. "Gene therapy for ischemic heart disease." Journal of molecular and cellular cardiology 50.5 (2011): 742-750.

[21] Lin, Hua, et al. "Expression of recombinant genes in myocardium in vivo after direct injection of DNA." Circulation 82.6 (1990): 2217-2221.

[22] Losordo, Douglas W., et al. "Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia." Circulation 105.17 (2002): 2012-2018.

[23] Nishida, Takahiro, et al. "Adenovirus-mediated local expression of human tissue factor pathway inhibitor eliminates shear stress–induced recurrent thrombosis in the injured carotid artery of the rabbit." Circulation research 84.12 (1999): 1446-1452..

[24] Pleger, Sven T., et al. "Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model." Science translational medicine 3.92 (2011): 92ra64-92ra64.

[25] Rajagopalan, Sanjay, et al. "Clinical Investigation and Reports-Regional Angiogenesis With Vascular Endothelial Growth Factor in Peripheral Arterial Disease: A Phase II Randomized, Double-Blind, Controlled Study of Adenoviral." Circulation-Hagertown 108.16 (2003): 1933-1938.

[26] Reilly, John P., et al. "Long‐term (2‐year) clinical events following transthoracic intramyocardial gene transfer of VEGF‐2 in no‐option patients." Journal of interventional cardiology 18.1 (2005): 2731.

[27] Rosengart, Todd K., et al. "Long-term follow-up assessment of a phase 1 trial of angiogenic gene therapy using direct intramyocardial administration of an adenoviral vector expressing the VEGF121 cDNA for the treatment of diffuse coronary artery disease." Human gene therapy 24.2 (2013): 203-208.

[28] Rosengart, Todd K., et al. "Angiogenesis gene therapy: phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease." Circulation 100.5 (1999): 468-474.

www.irjmets.com

@International Research Journal of Modernization in Engineering, Technology and Science

[214]

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume:02/Issue:09/September-2020

Impact Factor- 5.354

www.irjmets.com

[29] Rouis, M., et al. "Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions in apolipoprotein E–deficient mice." Circulation 100.5 (1999): 533540.

[30] Sarmiento, Nayade, and Selena Tinoco. "Bionatura Conference Series Vol 2. No 1. 2019."

[31] Stewart, D. J., et al. "Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF 121 (AdVEGF121) versus maximum medical treatment." Gene therapy 13.21 (2006): 1503-1511.

[32] Stewart, D. J., et al. "Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF 121 (AdVEGF121) versus maximum medical treatment." Gene therapy 13.21 (2006): 1503-1511.

[33] Stewart, Duncan J., et al. "VEGF gene therapy fails to improve perfusion of ischemic myocardium in patients with advanced coronary disease: results of the NORTHERN trial." Molecular Therapy 17.6 (2009): 1109-1115.

[34] Stewart, Duncan J., et al. "VEGF gene therapy fails to improve perfusion of ischemic myocardium in patients with advanced coronary disease: results of the NORTHERN trial." Molecular Therapy 17.6 (2009): 1109-1115.

[35] Su, Hua, Ronghua Lu, and Yuet Wai Kan. "Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart." Proceedings of the National Academy of Sciences 97.25 (2000): 13801-13806.

[36] Tammela, Tuomas, et al. "The biology of vascular endothelial growth factors." Cardiovascular research 65.3 (2005): 550-563.

[37] Tio, Rene A., et al. "Intramyocardial gene therapy with naked DNA encoding vascular endothelial growth factor improves collateral flow to ischemic myocardium." Human gene therapy 10.18 (1999): 2953-2960.

[38] Vassalli, Giuseppe, and David A. Dichek. "Gene therapy for arterial thrombosis." Cardiovascular research 35.3 (1997): 459-469.

[39] Waugh, J. M., et al. "Local overexpression of thrombomodulin for in vivo prevention of arterial thrombosis in a rabbit model." Circulation research 84.1 (1999): 84-92.

[40] Ylä-Herttuala, Seppo. "Endgame: glybera finally recommended for approval as the first gene therapy drug in the European union." Molecular Therapy 20.10 (2012): 1831-1832.

[41] Ylä-Herttuala, Seppo. "Gene therapy for heart failure: back to the bench." Molecular Therapy 23.10 (2015): 1551-1552.

[42] Ylä-Herttuala, Seppo, and Andrew H. Baker. "Cardiovascular gene therapy: past, present, and future." Molecular Therapy 25.5 (2017): 1095-1106.

[43] Zhang, Yue, et al. "Targeted delivery of human VEGF gene via complexes of magnetic nanoparticleadenoviral vectors enhanced cardiac regeneration." PLoS one 7.7 (2012): e39490.

[44] Zoldhelyi, Pierre, et al. "Thromboresistance of balloon-injured porcine carotid arteries after local gene transfer of human tissue factor pathway inhibitor." Circulation 101.3 (2000): 289-295.

[45] Zoldhelyi, Pierre, et al. "Prevention of arterial thrombosis by adenovirus-mediated transfer of cyclooxygenase gene." Circulation 93.1 (1996): 10-17.

www.irjmets.com

@International Research Journal of Modernization in Engineering, Technology and Science

[215]