AN EXCITING bit of news was announced at the last annual meeting of the Association for Research in Vision and Ophthalmology (ARVO) in Florida three weeks ago. Three dogs born blind were given the gift of sight for the first time after scientists injected a chosen gene into their eyes. The dogs were born blind because they lacked the gene that produces the protein called RPE65, which is necessary to enable their retinae to perceive light and register images. The disease that suffer from is called Leber Congenital Amaurosis (LCA), which is belongs to a class of several incurable forms of blindness that are collectively called as Retinitis Pigmentosa or RP. The team of biologists and ophthalmic surgeons from the University of Pennsylvania gave a single injection of the gene for RPE65 to the dogs by reaching from the front of the eye into the sub retinal space. They then monitored the progress of the treatment through electrical signals generated by the retina, called ERG's. They also video graphed the activities of the dogs as they were asked to navigate through a cluttered room. Since the dogs were given the gene therapy only in their right eyes, with the left eyes left untreated, restoration of light occurred only in the right eye. This was obvious both in the patterns and in the way the dogs avoided bumping into objects on the right side and turned their heads to see an observer standing behind them. It has been nine months since they got their single shot of the gene and all is well so far.

The presentation of this paper and the video expectedly caused a flutter a the ARVO meeting. While talking to the team that did the work, I found them to be appropriately cautious and conservative about their findings. As Dr. Albert Maguire, the surgeon who presented the work, said: the surgeons are ready and it the biology that has to be refined. They want to make sure that there is no toxicity or side effects. They want to know how long the effect will last, whether more shorts are necessary, when is the best time or age of the animal to take the therapy, and of course how to translate this into therapy for humans.

The people at the Foundation for Retina Research, started by David and Betsy Brint of Illinois (show son Alan is born blind with LCA), who supported this research in part, are "bursting at the seams", as reported by the Washington Post. (Incidentally, it is a particularly noble practice of the American society to encourage and fund research and treatment modes in diseases and afflictions of this kind, on the idea that such personal tragedies need to be remedied at large. How I wish we in India learn to emulate this practice).

The immediate question that springs in everyone's mind is whether the same treatment can be tried in humans. As many as 10,000 Americans are estimated to be born blind with LCA. In societies where intra-community and consanguineous marriages are practiced, such as in the Middle East and certain parts of the Indian subcontinent, the prevalence is expected to be higher, since LCA is an inheritable form of retinal blindness. The word of caution that Dr. Maguire used are appropriate here. Gene therapy has already been tried on some humans for other diseases. There has been some success in treating three babies (1 to 11 month old) who suffer from a fatal form of severe combined immunodeficiency syndrome or SCID. Dr. Alain Fischer and colleagues in Paris took blood stem cells from these infants and introduced into them the gene for a factor that goes to correct the deficiency, and put them back in the babies. It has been close to two years now and the children are doing well. On the other hand, however, is the tragic case of a 18-year old boy called Jesse Gelsinger, who died in a Phase I safety clinical trial of a gene therapy regimen involving the introduction of a gene called OTC, packaged in a virus used as the delivery vehicle or the vector. Jesse's death was directly attributed to the viral vector. Another patient who received the same treatment in the same trial, however, did not suffer any serious consequences. Following the death, gene therapy trials were stopped in the US, and are undergoing closer scrutiny, and several expert groups are refining and defining new measures and protocols.

What is gene therapy? An excellent primer on the basis of the method and a state-of-the-art review was given a few weeks ago by Dr. Inder M. Verma of the Salk Institute, San Diego, California when he delivered the Foundation Day Lecture of the Centre for DNA Finger printing and Diagnostics (CDFD), Hyderabad. Dr. Verma is a pioneer in the field of gene therapy, and has several other firsts to his credit. He was among the first to show that cancer is a disease of the genes, and to identify and characterize one such "oncogene" associated with the disease. Over a dozen years ago, he introduced certain types of viruses as candidates to deliver therapeutic genes. He is also the first holder of the G.N. Ramachandran Chair of biotechnology, instituted by the DBT of India. In the CDFD lecture, Verma discussed the possibilities, trials, tribulations and also some tentative triumphs in the dawn of early light in the field of gene therapy. He has also published a topical account of the field along with Nikunj Somia, in the November 2000 issue of Nature Reviews: Genetics. As Somia and Verma point out, the basic concept of gene therapy is disarmingly simple introduce the gene, and its products should cure or slow down the progression of a disease. It involves three steps. First is the mode of introduction. This is done by taking the desired gene and putting it in a "vector" or vehicle that carries it to the site. The second step involves the "expression" of the gene at the desired locale in high titre, i.e., at significant levels so that it does its function well. Thirdly, the body should not mount a rejection or immune reaction against the new gene or its products, or be otherwise adversely affected.

The therapeutic gene can be delivered using special lipid envelopes called liposomes, or shooting it straight into the target organ using a cellular scale "shotgun", or even straight injection of the gene into the tissue, particularly in some muscles. But the gene transfer efficiency in these instances is low. Also the expression of the introduced gene is transient. Since what is needed is sustained and high level expression, people have found viruses to be an attractive vector to deliver the transgene, since they easily get into cells and multiply there, and thus provide multiple copies of the desired gene. Tissue or organ-homing viruses can be used, since they carry their target detection system on their outside surface or coat. The first thing to do is, of course, to tame the virus vector by removing or disabling the offensive genes responsible for its pathology. After all, what one wants is to used the virus to infect and multiply but not cause any harm to the hot tissue. Viruses, in general, are light travelers; they carry in their genome just the basic set of genes needed to recognize and enter the host, multiply there using the host machinery, reassemble and exit in order to infect newer hosts. What the gene therapists do is thus to prune or edit the viral genome to the desired harmless level, and stitch in the therapeutic gene. This man-made construct is then allowed to infect, or transducer as the word goes, a target cell where it multiplies and buds off this packaging cell. We now have a large number of copies of the viral vector carrying the therapeutic gene.

Of all the viruses that can be used as vectors, retroviruses such as the AIDS virus are the most popular. They have a very small genome with but a handful of genes. Some of them, called lentiviruses, are able to transduce even non-dividing cells of the body such as those of the eye and the brain. In addition, they produce high titre, or measurable amounts of products, on a commercial scale. Construction of such custom-made viral vectors has been a flourishing R & D activity, and several such vectors based on retroviruses, lentiviruses, adenoviruses and adeno-associated viruses are available. Come to think of it, what a wonderful way to take these "bad guys" and tame them into useful cargo carriers!

Once the viral vectors are delivered into the desired body tissue where the therapeutic correction is needed, they do their job quite well. In other words, the second step of the expression of the added gene in the host tissue has not caused any serious side effects of problems. The French group delivered the therapeutic gene using a mouse leukemia virus vector when they treated the SCID babies. The Pennsylvania group delivered the RPE 65 gene to the LCA dogs using an adeno-associated virus, while Jesse Gelsinger and his cohorts were delivered the OTC gene through an adenovirus. In each of these instances, the expression of the desired gene product was no problem and the defect in the patient was rectified.

What then are the difficulties facing gene therapy technology now? The biggest challenge facing all viral vectors is the immune response of the host. The host can react and defend itself at the cellular level, by generating cell-killer T cells, and eliminate the transduced cells and nullify the effect of gene therapy. The other defense mechanism operated at the humeral level is by generating antibodies against the viral proteins, which would act against repeat administration of the vector. Current work in the area is focused on reducing the immunogenicity of the viral vectors. Biochemists are also trying out efficient non-viral delivery vehicles, which may do away with these virus-associated hurdles. Somia and Verma discuss the various strategies that are being tried to circumvent this problem.

With the French success with SCID children and the more recent American success with LCA dogs, hopes on gene therapy will rise. Gene therapy is a recent technique that involves input from the geneticist, the virologist, the molecular and cell biologist, the clinician and also the ethicist. It has been tried for single gene defects, and people will try to pack multiple genes also into vectors. It is not yet good for all genetic diseases, for example those afflicted with trisomy, but I would not be surprised if someone comes out with a specific sub-chromosomal or gene silencer. As Verma points out, the science of gene therapy has many hurdles ahead, but they are surmountable.

The Hindu Businessline
Genetics
May 24, 2001