Gene Therapy In India

Published Date: 02 Nov 2017

Globally, the sufferers of genetic disorders are enormous in number. Cure for genetic diseases was sci-fi before the first success story of gene therapy. Gene therapy offers fantabulous potential to treat and cure many genetic diseases. Gene therapy is a technique of replacing the defective genes with healthy genes or repairing the imperfect genes in order to restore the lost functions of the genes. Gene therapy showed enormous success in treating monogenic disorders like Severe Combined Immunodeficiency (SCID), Parkinson’s disease, melanoma, etc which progressed to various phases of clinical trials. United States and Europe are pioneers of gene therapy and clinical trials are being carried out for more than two decades in these countries. The discovery of viral vectors is a turning point in gene therapy research. Use of viral vectors enhanced the success rate of clinical trials world wide. Viral vector mediated gene therapy is extremely efficient and is being used by the scientists to drive expression of many genes. Recently, many gene therapy labs have come up in India with the support of Indian government to develop expertise in treating various diseases by gene therapy. However, gene therapy research in India is still in infancy which requires lot of knowledge, training, awareness and integration between the researchers and clinicians in order to reach clinical trial stage. This review summarizes the applications, limitations and scope of gene therapy.

Applications of Gene Therapy

Out of the several applications of gene therapy, the following represent major ones.

Gene therapy is most approachable for single gene disorders as targeting one gene is relatively simpler with the currently available gene delivery technology and also due to the relative ease in monitoring the effects of monogenic gene therapy. In fact, X-linked severe combined immunodeficiency (X-SCID) was the first successfully treated monogenic disease by gene therapy.

Ocular gene therapy is highly promising to treat congenital optic and optic nerve disorders. As eye being a small organ, it is possible to transfect a large number of ocular cells efficiently. Ocular gene therapy is at its lead as it proved success by restoring vision in 3 young patients suffering from Leber’s congenital amaurosis (1). Other optic nerve diseases like glaucoma and Leber’s hereditary optic neuropathy (LHON) are also being studied for gene therapy which already showed success in animal models.

Gene therapy has fullest application in treating few neurological diseases related to central nervous system. The available novel methods of gene therapy increase the possibilities of successfully treating genetic disorders like Parkinson’s disease. Till date several gene therapy clinical trials specifically phase I and phase II trials using both adeno-associated viral vectors and lentiviral vectors for Parkinson’s disease have been recorded testifying the efficiency of gene therapy (2,3).

Gene therapy has vast applications in treating a range of cancers. Till date, around 64.3 % of the total clinical trials recorded worldwide accounts for cancer gene therapy. The genes in the tumour cells can be altered or replaced by healthy genes in order to correct the defect or to improve the patient’s immune system to fight against other tumour cells. The cure of melanoma in 2 patients is substantiation that gene therapy is an excellent treatment option to cure cancers (4). Apart from correcting the defective genes, scientists are even investigating to insert suicidal genes like the herpes simplex virus thymidine kinase gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, etc which have potential to destroy the other cancer cells and diseased proliferating cells. This is a very significant strategy to arrest the uncontrollable division of cancer cells which would even offer eradication of several types of cancers.

Other genetic disorders like Cystic fibrosis, Chronic Granulomatus Disorder (CGD), Hemophilia, Leukocyte Adhesion Deficiency (LAD) and acquired diseases like diabetes, HIV, hepatitis and several cardiovascular diseases are being studied by scientists to cure them by gene therapy.

Some life threatening genetic diseases can be rectified before birth of the baby by fetal gene therapy termed as in utero gene therapy or prenatal gene therapy. The treatment aims at correcting the genetic defect in the fetus itself to prevent the occurrence of life threatening diseases at an early onset. In utero gene therapy was successfully carried out in animal models of human genetic diseases (5).

Exploration of solutions to overcome the problems involved in gene therapy

Human gene therapy has quite a few limitations which are extending as problems being faced by the researchers during gene therapy preclinical and clinical trials. Some of these problems are highly risky which might lead to adverse effects in the patients. With the significant advancement in genetic engineering, molecular biology, vector biology and technology, scientists are trying to unravel the possibilities to overcome the roadblocks in current gene therapy procedures.

Achieving long term benefit from the therapeutic gene is a difficult task due to the inbuilt rapid differentiating nature of the cells. Multiple doses of the DNA have to be administered which is practically not easy. One way to accomplish persistent expression of the transgene is to choose efficient vector system which can integrate the curative gene into the host genome at a single dose of administration. For example use of lentiviruses as gene cloning vectors showed persistent expression of the candidate gene after 30 months of post treatment follow-up (6).

Gene therapy for multigenic diseases like cancer, diabetes, Alzheimer’s disease and some cardiovascular diseases is complicated compared to monogenic disorders as targeting and correcting several genes is difficult. However, few studies evident that it is not impossible to treat multigenic disorders with development of modified viral vectors capable of expressing more than one therapeutic gene.

The inserted genes sometimes fail to express after introduction in the host due to gene silencing. Some transgenes silence after expressing for certain period of time. There are gene therapy studies which showed high expression of the therapeutic gene in vitro and the transgene failed to express in animal models subjected to autologous ex-vivo gene therapy (7). Various factors like DNA methylation, histone modification, binding of various transcription factors, transgene integration site, promoter and vector properties also might account for the transgene silencing. Employing gene therapy strategies for site-specific transgene integration, use of tissue-specific and ubiquitous promoters for driving the gene expression, addition of regulatory elements to the cloning vector for stable transcriptional activity of the transgene, use of low immunogenic vectors, etc might help reducing gene silencing.

Viral vectors are the most powerful gene cloning vehicles but there are some serious practical problems involved in using viral vectors for human gene therapy. The viruses might replicate inside the host’s body which is undesirable and a great threat to the host. Re-engineering the retroviral genome by removing of viral genes required for replication in the host and splitting the viral genome into helper plasmids led to the development of various generations of retroviral vectors termed as replication competent viral vectors and self-inactivating viral vectors which ensure safety and stable gene expression.

Genotoxicity resultant from random integration of the viral genes into the host genome is a potentially dangerous condition. This is a critical problem being encountered in gene therapy in which the viruses sometimes target wrong cells and insert themselves at wrong place leading to devastating effects. This might lead to oncogenesis as in the case of two X-linked severe combined immunodeficiency (X-SCID) patients who developed leukemia like disease on receiving retrovirus mediated gene therapy (8). The integration site analysis of the viral genome even before using the vectors for invivo gene therapy might be of great benefit to reduce the risk of oncogenesis.

Few viral vectors might trigger host immune responses that lead to multiple organ failure and other life threatening complications. Immunogenicity of the viral vectors is often a hurdle in gene therapy due to the highly fatal consequences. For instance, adeno viral vectors are very efficient vectors as they can transduce both proliferating cells and mitotically inactive cells but a major disadvantage is their immunogenicity. However, scientists are focusing on application of immunosuppressive drugs to reduce or eradicate immune responses in the patients treated by viral vectors and also development of adenoviral vectors which are less immunogenic by modifying the viral genome.

Some viral vectors like adeno associated viral vectors are very efficient but have limited packaging capacity. This is due to the small genome size of the vector and cannot accommodate larger transgenes. The only alternative to overcome this problem is to choose vectors into which larger transgene could be cloned and expressed.

Setbacks of gene therapy

The medicine of future had two serious failures in the past which hindered many gene therapy studies and clinical trials. In 1999, an eighteen years old boy called Jesse Gelsinger who was suffering from ornithine transcarbamylase deficiency, an X-linked genetic disease of the liver was subjected to gene therapy treatment using adeno virus vector carrying human ornithine transcarbamylase cDNA. He died after 98 hours of injection of the viral vector with severe immune response which led to multiple organ failure due to the use of first generation viral vectors (9). This was an adverse event which attributed the researchers to look at the safety of using viral vectors for human gene therapy.

In another gene therapy clinical trial, 2 out of 10 X-linked severe combined immunodeficiency (X-SCID) patients who were treated with a retroviral vector developed uncontrolled exponential clonal proliferation which gave rise to leukemia like disease within a year of treatment. This was due to the integration of retroviral vector in the proximity to a proto-oncogene promoter region called as LMO2 (8). This resulted in the temporary halt of many gene therapy clinical trials throughout the globe especially the trails involving viral vectors by the US Food and Drug Administration (FDA).

Recent successful clinical trials worldwide

Though gene therapy had some initial setbacks, it proved success in some of the recent clinical trials which paves path to treat and cure many genetic diseases.

For the first time in the history of genetic retinal diseases, gene therapy restored vision in 3 young patients with Leber’s congenital amaurosis, a common cause of blindness in infants and children. This was reported in the year 2009 after a year follow-up after the correction of mutation in the RPE65 gene encoding retinal pigment epithelium– specific 65-kD protein (RPE65) (1). The vector used to carry the gene was recombinant adeno-associated virus serotype 2 (rAAV2) vectors which was infused by subretinal injection. This study showed the efficacy of gene therapy using viral vector and proved safe even after 2 years of actual treatment.

An interesting study was published by Cartier et al in 2009.A severe brain demyelinating disease, X-linked adrenoleukodystrophy (ALD) was successfully treated by hematopoietic stem cell gene therapy. Two ALD patients were genetically corrected by ex vivo autologous gene therapy in which a lentiviral vector encoding wild-type ABCD1 gene was used. Both the patients exhibited successful results 14 to 16 months post treatment. The progressive cerebral deamyelination stopped and polyclonal reconstitution was observed with 9 to 14% of granulocytes, monocytes, T and B lymphocytes expressing the therapeutic protein. Till date, no side effects have been reported in both the patients (6).

In 2009 another promising clinical trial was carried out by Aiuti et al in 10 severe combined immunodeficiency (SCID) patients with adenosine deaminase (ADA) deficiency who lack HLA identical sibling donor. These patients were infused with autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene after nonmyeloablative conditioning. All the patients are alive after a follow up of 4 years and no adverse events were reported after the treatment except in 3 patients. In 9 patients the immune function was restored and protection against infections was also seen (10).

In 2006, Rosenberg’s team reported regression of metastatic melanoma by autologous gene therapy in 2 out of 15 cancer patients treated. A retroviral vector encoding T cell receptor was used as cloning vehicle to carry the therapeutic gene. High and sustained levels of engineered lymphocytes were observed in both the patients (4). For the first time, this group showed effective and potential cure of melanoma using autologous gene therapy. This is an amazing breakthrough in the field of cancer gene therapy as the deadly disease which had no cure is now curable by gene therapy.

Where Gene Therapy is, in India

USA is the first country to attempt gene therapy trial in the year 1980.Since then, till 2012, about 1843 gene therapy clinical trials have been recorded and progressed from preclinical level to clinical study level, worldwide. Countries like US, UK, Japan, etc. are proving their best in gene therapy clinical trials. However, in developing countries, gene therapy is still in its infancy. India has reached to a considerable level in gene therapy research. This can be proved by the various research projects being carried out currently and various new gene therapy labs that have come up in our country. Till late 1990s, there was not a single gene therapy laboratory or gene therapy research project in India. After 1998, a good number of gene therapy laboratories have been started in different parts of India. Between 2000 and 2012, there was an emergent enhancement in the number of gene therapy laboratories. Nearly 5 new labs have come up with a wide range of research facilities to carryout the preclinical gene therapy research.

Advanced Centre for Treatment, Research and Education for Cancer (ACTREC), Mumbai is the first institution to start gene therapy research in India in 1998. Dr.Rita Mulherkar’s group from ACTREC is actively carrying out gene therapy for head and neck cancer using synthetic vectors. In 2005, a bio company named Actis Biologics Private Ltd.(ABPL) in Mumbai started a gene therapy lab. ABPL is currently working on the introduction of Gene MSP36 into cancer cells to cause them to die by production of a protein, which will inhibit the rapid multiplication of the cells and their metastisis. Two scientists from Center for stem cell research (CSCR) in Christian Medical College, Vellore have established their laboratories in the year 2010 and are currently focusing on extensive gene therapy research. Dr Jayandharan G Rao from CSCR is carrying out studies on Adeno-associated virus is its application to translational gene therapy in therapeutic models such as pre-clinical animal models of hemophilia. Another scientist, Dr Sanjay Kumar from the same center is currently involved in gene therapy using Adeno-associated viral vectors. His research focuses on use of genetically-modified stem cells in experimental gene therapies. His research interests are tissue-specific targeting of mesenchymal stem cells (MSCs), ex-vivo modification of MSCs using adeno-associated viral vectors for gene therapy applications and studying biological characteristics of subset of stem/progenitors in spinal cord and bone marrow. He is simultaneously investigating molecular interplay between pathologic stem cells with regard to their interaction with the micro-environmental cues.Dr Rupesh Dash from Institute of Life Sciences, Bhubaneswar began cancer gene therapy project in the year 2011. He works on systemic and targeted gene therapy for cancer, targeting anti-apoptotic Bcl-2 family members for effective therapy of oral squamous carcinomas. A new gene therapy lab has been set up in 2011 at Vellore Institute of Technology (VIT), Vellore with Dr Everette Jacob Remington N as principle investigator for the project Gene therapy for leukocyte adhesion deficiency using lentiviral vectors. This project aims at ex-vivo modification of hematopoietic stem cells using lentiviral vectors and re-infuse into murine LAD models

Apart from all the well established labs mentioned above, a handful of new labs are coming up in various parts of our country namely in Bangalore, Pune and Kolkatta. This is a clear indication of India’s interest and progression in gene therapy research.

Scope:

Genetic diseases were once incurable till gene therapy came in to light. World Health Organization reports that over 10,000 different genetic diseases specifically monogenic disorders are affecting mankind globally. Treatments are available to cure the phenotypic effects and symptoms of the genetic diseases but there are no definitive treatment options which could completely eradicate many genetic ailments. Correcting the genetic defect at its root level rather than treating the symptoms would be a permanent solution for many life threatening diseases and abnormalities. Gene therapy already showed success in treating genetic diseases and even one form of cancer. It is obvious that gene therapy is not restricted to monogenic disorders; it could be used to treat several fatal diseases like cancer, HIV and other infectious diseases. With the complete knowledge about the disease biology, advancement in vector technology and integration of scientific knowledge, gene therapy could be a magnificent treatment option and hence it is called "the medicine of future".

CONCLUSION:

The number of gene therapy clinical trials for a range of diseases is escalating every year worldwide. American and European continents are excelling in gene based therapies which is evident from the progress of their gene therapy research from experimental level to various phases of clinical trials. All the successful clinical trials reported till date are from USA and Europe. There is no doubt that even Asia is advancing in the development of gene therapy strategies for various diseases. Among all the Asian countries, China has highest number of gene therapy laboratories/projects (Fig. 1). India has six gene therapy laboratories altogether out of which five have come up only after 2000 (Fig. 2). Indian government is now trying its best to improve gene therapy research in India by providing extensive financial support to knowledgeable, efficient and experienced scientists through various funding agencies like Department of Biotechnology (DBT), Department of Science and Technology (DST), Indian Council of Medical Research (ICMR), etc. With the same level of financial support in future, India is sure to reach clinical trail stage with regard to gene therapy. Over time, gene therapy will be an incredibly effective treatment in the modern medicine era which could completely stamp out genetic and non genetic diseases.

Figure 1: Number of gene therapy laboratories/projects in Asian continent.

Figure 2: Graphical representation of the increase in number of gene therapy labs in India between 1990 and 2012.