Gene Therapy : Dr. Anil Neelakantan ( view bio )

Kakes, as I had promised, I would post something about gene therapy. I have read about this during house surgeoncy and now it's been at least a year. Therefore, I hope you would pardon me if I make any mistakes in my attempt to sketch out the frontiers of gene therapy.

Though the idea of genetic manipulation has not been novel to the scientific world, the issues involved and the lack of a flawless technique to make this a reality were always hindrances to its progress. It goes without saying that until recently there was no known way of curing a genetic disease. So the natural consequence of the search for treatment of such illnesses culminated in the development of gene therapy. Which are the diseases that are could be cured by this method? It is well known that transfer of genes is a tedious process and is potentially hazardous. Hence, the candidates now are those diseases that could be treated with minimal manipulation. Diseases where multiple genes are involved are currently out of the fray. The first disease to be studied was cystic fibrosis (located on the 7th chromosome -if I remember it correctly). Then came the diseases like Parkinsonism, Familial Hypercholesterolemia, and immunological disorders of adenosine deaminase deficiency (KKV, now I know that SCID is one of them). Later the concept of gene therapy was totally changed when they found that it could not only be used to treat inherited disorders but also malignancies that may not even have a genetic cause! (I will give an example for this later) Recently, Duchenne's Muscular dystrophy was also cured by this method although it is known to be caused by more than two genetic defects.

So now we know how these diseases were chosen. I would like to describe how could we go about doing this. Gene therapy can be can be basically divided into two varieties viz. Vector mediated and Non-vector mediated. In vector mediated an organism, usually a virus, delivers the normal gene to the target. While in non-vector mediated method, a direct method of transfer is used. In vector mediated gene transfer, the virus is stripped off the genes responsible for inducing disease while the genes responsible for infectivity are retained. Hence we get a live infective virus that is 'castrated' (they cannot produce illness). The genetic make up of any living organism has a memory ceiling i.e. the maximum number of codons that it can withstand without being unstable. Hence we can only insert a gene that has a size almost equal to that of the deleted genes. Say 120kb (120kilo base pairs). This helps us to determine the gene (and hence the disease) that could be carried by a specific vector (depending on how much could be deleted from the vector). Once the gene-stripped viruses are ready, we culture them in an appropriate medium. The medium would have viruses with plasmids that contain the stripped off genes so that they would produce the proteins that are essential for the virus to grow. Now the viruses that do not contain the plasmids are separated and prepared for the invitro gene transfer (into the virus) so that we would get fully functional vectors ready to do their work. The gene to be delivered is isolated from a normal human and amplified using PCR. Two sticky sets of codons are attached to both ends of this gene so that they could be inserted into the appropriate areas in the viral DNA. The genes are transferred in two ways. Either using bacteriophage where it would be incorporated in to the vector DNA or by just placing the virus in a medium that contains a large quantity of the sticky therapeutic gene so that it would be taken into the virus by pyknosis and would form a plasmid in the cytoplasm of the vector. These are then passed through a series of stringent tests to ensure that there would not be any infective virus in the lot. The current purification techniques have been able to do this to a purification level of 99.9%. But even 0.1% is dangerous. This is then injected into the site where gene transfer is required (like liver, brain etc). Another method tried was to identify a unique protein on the surface of the human cell into which the transfer has to occur and attach a gene into the vector that would produce the complementary protein structure so that it would specifically attach onto that cell type with just a systemic administration (like an intravenous injection.). The viral DNA would normally become part of the host DNA upon infection. At the same time, it is not capable of producing any disease. So the ultimate result would be the incorporation of the viral junk DNA along with the required gene of therapeutic interest. And the cell would start transcribing it . We can introduce the p 53 gene to prevent cancers. It could also be used to insert deficient genes. It would also help to incorporate a terminator codon onto the 5' end of a malicious gene in the human cell so that the cell would not transcribe it. The possibilities are enormous. The greatest of them would be treating cancer, which does not have a genetic basis! The viruses used for the purpose are Adeno virus, Herpes virus and retroviruses.

Glioblastoma multiforme and other gliomas are often fatal. No consistent gene defect has been attributed to this. However, even this disease can be cured by gene therapy. It is well known that TK gene of humans is different from that of viruses. Acyclovir specifically inhibits the Herpes TK gene and hence it can function as an antiviral agent. Herpes virus in its natural course is neurotropic. Hence, by introducing Herpes TK gene into the malignant tumor we can get them to produce viral TK. When this is combined with the incorporation of a terminator codon prior to the human TK gene, the only source of TK for those cells would be the Viral gene. When delivered to the tumor directly, by an intra lesional injection, the net result would be a set of viral TK producing malignant cells. Now treat this patient with acyclovir. We can convert this tumor mass into a necrosed area in the brain. This liquifactive necrosis would result in the formation of a brain abscess, which has a better outcome than a malignant glial tumor. And the patient would then be subjected to an intensive antibiotic therapy along with adjunct measures like drainage once it is certain that there are no residual tumor cells (for fear of deposition in the needle track). Thus, we could use an antiviral agent as an anti-cancer drug and treat a malignancy, which has no direct genetic etiology.

In the non-vector method liposome mediated transfer of genes or intracellular injections of genetic material using micro-pipette are resorted to. This is tried in Duchenne's dystrophy where intramuscular injections of genes are given.

In AIDS virus, the essential genes are env, pol and gag These are essential for the virus to multiply in the CD4+ cells. If we could have a vector mediated site specific terminator codon gene transfer, we could prevent the multiplication of virus in these cells. Hence, it could possibly be a method of vaccination against AIDS (because the presence of these genes before hand would prevent the successful multiplication of retroviruses on entry).

Gene therapy is involved with a lot of complications ranging from mild rhinitis to severe anaphylactic reaction to any of the components used in the drug. In addition, the presence of live virus contaminant could be very dangerous, especially in case of retroviral vectors. The 'mal-union' of the genes with the host DNA could have devastating consequences. So gene therapy is a method that is yet to be perfected. It has to successfully pass the current stage of Phase 2 clinical trial in order to be available to the common man. As usual, we should expect that the initial expense would be exorbitant and would be unaffordable to the poor (due to patenting issues). Nevertheless, ultimately it has to be in vogue.

I hope I could throw some light into what gene therapy is all about. Please pardon me for any mistakes, as it is quite long since I read the topic.

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