Strategies And Vectors For Gene Therapy: Its Prospective Therapeutic Attributes Against Restenosis

Abstract

Gene therapy is seen as one of the upcoming technologies not only against diseases which have monogenetic etiology, but also against complex diseases such as cancer and cardiovascular disorders. Amongst the cardiovascular disorders, restenosis is one of many disorders which has seen a major increase in the clinical trials, using gene therapy, in recent years. Restenosis, which is simply reoccurrence of stenosis, is seen mainly post surgically in an artery or blood vessel which had been unblocked. Importantly, even though stents have been introduced to prevent restenosis to occur post surgically, the effect seems to be limited to decreasing the statistical rate , and restenosis still persists as a problem for which a definite solution or remedy, acting on the very roots of its pathogenesis, is the need of the hour. Gene therapy, transfer of a healthy gene for curing a disorder, seems to a promising modality for the purpose. To meet this end a definite strategy, an appropriate vector and target for efficient and persistent expression of the healthy gene in the desired or localized area, is what will make gene therapy against restenosis more effective.

Restenosis, Gene therapy, Vector, Remodeling

Introduction
About 40-50% of vessels undergo Restenosis after Coronary Artery Bypass Graft (CABG) or Percutaneous Coronary Angioplasty (PTCA), and in occasions where traditional stents are used (.i.e. when the diameter of the vessel is > 3mm in diameter) this rate is reduced to 20% -30% (1). This loss of lumen in a previously operated / dilated artery which results in poor vascular patency is due to increase in number of intimal (inner layer of the vessel) cells, known as neointimal hyperplasia. Neointimal hyperplasia along with constrictive remodeling are the two phenomenons responsible for restenosis, and in both, extra cellular matrix (ECM) accumulation is the causative factor (90% of the bulk of neointima comprises of ECM). Constrictive remodeling is said to be the major cause for this luminal loss, especially vessels which have been dilated due to atherosclerosis as the primary cause (1). This remodeling can be prevented by transferring a healthy gene, into the patient’s body, which is thought to be playing a pivotal role in its formation. One of the key challenges at present is finding the appropriate vector for delivering a healthy gene or a cocktail of genes (multigenic approach) in the target tissue. Another aspect that needs to be considered is the duration of the gene expression, post gene delivery, by the vector.

Material and Methods

Strategies For Gene Delivery
Cardiovascular diseases can either be inherited or acquired, and each type needs to be dealt with a different strategy (2) (Table/Fig 1) .There are basically two strategies used for the gene delivery, namely in-vivo and ex-vivo gene delivery. When therapeutic or desired genes are delivered inside the body then it is known as in-vivo gene delivery, if the cells are removed from the body and the therapeutic genes then transferred into the cells, it is termed as ex-vivo gene delivery. To achieve delivery of the desired gene or product in the target tissue certain steps need to be considered. Firstly, DNA (desired genes) must be delivered to the nucleus and secondly, the central dogma (DNARNA Protein (functional)) should follow. The first step can occur either in in-vivo or ex-vivo, but the second step always occurs within the body (in-vivo).

Ex-Vivo Gene Delivery
Ex-vivo gene delivery is a relatively simple method mainly used in vein graft failure. A good demonstration of its use was shown against familial hypercholesterolemia. The Kupffer cells (cells were taken out by partial hepatectomy) were cultured ex-vivo and then transduced with retrovirus containing the gene for LDL receptor, as a result there was decrease in cholesterol levels (3)(4). There are certain advantages with the ex-vivo gene delivery, for example, it has a high efficiency for gene transfer into the targeted cells, its specificity can be restricted to the desired cell type by careful optimization and designing and also the immune response to the vector transferring the gene is minimized as it is performed outside the host. The disadvantages for ex-vivo gene delivery may be due to the procedure involved, for example, the patient may have to undergo two invasive procedures one for the cell harvest and the other for the cell reintroduction after the transfer (like in the case of hepatocytes. (Table/Fig 2)

In-Vivo Gene Delivery
In In-vivo gene delivery there is only one procedure required, i.e., injection of the gene vector and there is no need of cell harvesting and reimplantation. Also any cell of the body organ is the potential target for the gene transfer. But there are some drawbacks with this method as well like, it will be difficult to reach to remote tissues like that of the myocardium or a narrow artery in which the vector may be washed away or the pathogenic mechanisms (e.g. ischemia) may occur before the transfer takes place. Also the systemic release of the vector would really be unavoidable and so optimization of the gene expression (localization) will also be hard to control. Also the vector may produce an immune response and result in a rejection to it, especially if the immune system has had a prior exposure to it. Keeping in mind the fact about the diversity of the cells as targets in our body, the vector system needs to be developed for individual applications (5) , and has a long way still to go to be able to give an efficient gene transfer at the same time meeting all the safety concerns.

Vectors For Gene Therapy
Vectors can be either non-viral or viral. At the moment, out of the two, non viral vectors are suggested to meet the properties of an ideal vector, simply because of it being nonpathogenic, more efficient in gene delivery and less immunogenic. Additionally, because the mechanisms, by which viral vectors work and can be controlled, requires a lot more research and better comprehension for them to be used therapeutically. But limitation of sustained gene expression by non-viral vectors needs to be addressed for it to make it to the clinical practice.

Non-Viral Vectors
This group of vectors consists of naked<

Discussion

The therapeutic attribute of gene therapy against various disorders, will require a well planned and systematic approach to be most effective. Many factors like etiology, choice of vector, mode of delivery of vector, choice of targets etc. all have to be carefully planned based on the merits of each case. Gene fingerprinting and pharmacogenomics may further accentuate its effectiveness.

At present optimization of various available vectors and search for new potential vectors is the area of focus in the field of gene therapy. More and more clinical trials are being initiated in this sector and many new strategies being tested. Many safety concerns and ethical issues have arisen with this methodology of treatment, and adverse effects like neoplasms, edema, immune responses, etc. have acted as a rate limiting step in the advancement of research in this field. But at the same time researches addressing these concerns have been very promising. Recent example being the discovery of a novel mechanism involving protein Hexon and a blood clotting enzyme ,Factor X by Dr. Baker’s group (Waddington et al) at the University of Glasgow. Mutations in the Hexon protein and pharmacological blockade of the interactions of these proteins blocked the gene transfer, suggesting the mechanism by which gene transfer takes place in case of fibre modified viral vectors (19).
This new fact can be used to design safer fiber modified vectors for gene delivery. Gene therapy could be the answer to many diseases, especially against Restenosis, for prevention of which today the most common tool are the stents. The incidence of Restenosis when no stent is used in 25-40% , but when a medicated stent is used this incidence can be brought down to 10-20%, which still is quite a considerable rate considering the number of CABGs carried out (20).

Gene therapy is one promising modality which can be combined with present modalities like coated stents ( which may no longer pose any threat of late and sudden occurrence of restenosis associated with it ) to fill this vacuum and act at the root level against restenosis .