Gene Therapy- A CONCEPT

If a child or an embryo is diagnosed to carry a defective gene leading to disability, one may like to correct this defect by any of the following three methods.

(i) by replacement of defective gene with a normal gene

(ii) by correcting the defective gene through gene targeting

(iii) by gene augmentation either through increasing the no. of copies of the gene or through a higher level of expression of the introduced gene .

Such a correction of a genetic defect is described as gene therapy.

Once the gene correction or gene augmentation has been achieved at the cellular level (in the cells obtained from the affected organ depending upon the disease), the modified cells can be implanted into a suitable region either in an organ of the patient or in the embryo. Direct delivery of the DNA (carried by the vector) into the living cells of the body has also been suggested in several cases, so that both in-vitro and in vivo introduction of corrected gene has been suggested.

Therefore, gene therapy can be used at two different levels : (i) embryo therapy, in which the genetic constitution of embryo at the post-zygotic level is altered, so that the inheritance will also be altered. (ii) Patient therapy, in which cells with healthy gene may be introduced in the affected tissue so that the healthy gene overcomes the defect without affecting the inheritance of the patient.

Maximum progress in the area of gene therapy has been achieved through the gene augmentation method involving vector mediated DNA delivery system.

During the last 5-10 years, it has become routine exercise to isolate any gene. This isolated Gene, may either be directly injected into the cell or be carried by a virus, to which it is linked by recombinant DNA technique. After entering the cell, the gene may become a part of nuclear DNA or remain free in cytoplasm like extra chromosomal DNA. However, in each case RNA is synthesized only at the rate of few copies per cell in comparison to normal cells where thousands copies are made.

Salient Features of Site Specific Gene Delivery System

1) This delivery vehicle (F-virosome) consisting of the envelope of Sendai virus containing only the fusion protein (F) and devoid of its own genetic material and other undesirable proteins, holds minimum risk of any side effects.

2) The F-virosomes containing the genes of interest (e.g. CAT and luciferase) specifically bind and fuse with liver parenchymal cells (both in vitro and in vivo) resulting in high efficiency gene transfer to the nucleus of these cells, bypassing the degradative endocytotic route.

3) A single intravenous administration of DNA loaded F-virosomes into Balb/c mice resulted in expression of CAT mRNA and the corresponding protein till four months post injection. The delivered DNA was found in the mouse chromosomes and persisted till two months.

4) No adverse immune side effects (antibodies against F protein) were observed in F-virosome injected mice.

5) Preliminary experiments have been done in delivering a therapeutic gene (bilirubin-UDP-glucuronosyl transferase, BUGT) into Gunn rats (lacking the BUGT gene showing high levels of serum bilirubin) through F-virosomes and found to be encouraging in terms of reduction of serum bilirubin levels.

6) US Patent has been granted (US Patent No.5,683, 866 dated November 4, 1997).

Novelty of F-Virosome Mediated Gene Delivery

1. Specific cytosolic delivery of genes and their expression in cells using Sendai viral envelope devoid of hemagglutinin-neuraminidase and its own genetic material thereby totally eliminating the possibility of any side effects. Sendai virus, which is the starting material for developing this delivery system, is known to be non pathogenic to humans.

2 Dual role (in binding and membrane fusion) of F protein of Sendai virus in gene delivery

3. Targeted delivery of genes into liver cells in culture using modified Sendai viral envelopes.

4. Considerably higher efficiency of gene delivery as compared to existing delivery systems such as liposomes and commercially available systems like lipofectin.

The genes delivered by this system remain intact and are able to express biological by active protein products.

Uses: Some of the Potential Diseases for which therapeutics can developed using this delivery system such as: Haemophilia, HBV, Diabetes, Cancer.