Basic Research

S. No.	Project	Principal Investigators	Duration
1	Preservation and use of adult stem cells and the use of biomaterial scaffolds for clinical applications	Dr. Virender Sangwan, Dr. Balasubramanian, Dr. Indumathi M, Dr. Jose Domingos dos Santos, Dr. Livia Santos, Dr. Ascensao Lopes & Dr. Ana Colette Mauricio	2011-2014
2	ProjectDerivation of retinal neurons from ciliary pigmented epithelium of the human eye	Dr. Indumathi M	2010-2013
3	Molecular mechanism of pathogenesis of glaucoma caused by mutation in optineurin	Dr. Subhabrata Chakrabarti , Dr. D Balasubramanian & Dr. Ghanshyam Swarup	2010-2013
4	Development of a synthetic biodegradable cell carrier membrane for the transplantation of cultured cells or freshly excised autologous tissue (limbal segments or oral mucosa) for diseases of the cornea	Dr. Virender Sangwan , Dr. D. Balasubramanian , Dr. Geeta K Vemuganti , Dr. Sheila MacNeil , Dr. Frederik Claeyssens , Dr. Anthony Ryan & Dr. Suzanne Bushnell-Watson	2010-2013
5	Investigation of epidemic keratoconjunctivitis in Orissa and Andhra Pradesh: Epidemiological profile, clinical spectrumm and molecular characterization of causative agents	Dr. Savitri Sharma, Dr. BNR Subudhi, Dr. Sujata Das, Dr. Srikant K Sahu & Dr. Somasheila Murthy	2010-2013
6	Program support on translational research on eye diseases	Project A: Dr Geeta K Vemuganti Project B: Dr Chitra Kannabiran Project C: Dr Inderjeet Kaur Project D: Dr Subhabrata Chakrabarti Project E: Prof D Balasubramanian	2007-12
7	Establishing a “Risk assessment chip” for the eye disease age related macular degeneration	Dr Subhabrata Chakrabarti Dr Inderjeet Kaur	2007-10
8	Cultivation and characterization of human lacrimal gland cells for potential clinical application for post radiotherapy xerophthalmia	Dr Geeta K Vemuganti Dr Santosh G Honavar Dr Vijay Anand P ReddyM	2008-12
9	Study of the role of vitreous liquefcation in age- related nuclear cataract	Prof D Balasubramanian Dr Pravin V Krishna	2009-11
10	An advanced surface for the cell therapy of limbal epithemium for ocular surface disease: Proof of concept and clinical trials	Dr Virender Sangwan Dr M Indumathi	2009-12
11	Developing a global blueprint for zero PCG	Dr Subhabrata Chakrabarti Prof Partha P Majumdar1 Prof Luba Kalaydijeva2	2008-11
12	Molecular characterization of nonsporulating flamentous fungi isolated from microbial Keratitis	Dr Ashok Kumar Reddy	2009-12
13	Evaluation and Characterization of cancer stem cells in Retinoblastoma tumor	Dr Geeta K Vemuganti	2009-10
14	Genetic and phenotypic comparison of primary congenital glaucoma in India and Brazil	Dr Subhabrata Chakrabarti & Dr Paulo Mello	2009-11
15	Central India Eye & Medical Study	Dr Subhabrata Chakrabarti & Dr. Jost Jonas
16	Genetic comparison of primary congenital glaucoma in India and Tunisia - Understanding the possible founder effects	Dr Subhabrata Chakrabarti & Prof G Fethi	2009-11
17	Generation of disease specific inducted pleuripotent stem cells from dermal fibroblasts of patients with retinal dystrophy	Dr Indumathi	2009-11
18	Champalimaud Translational Centre for Eye Research	Prof D Balasubramanian	2007-12

 

Details of the projects

1. Program support on translational research on eye diseases

India has about 15 million people who are blind. Of these, about 10 million are cataract blind, about 1.2 million blind due to uncorrected refractive errors, and another 1.0 million due to infection of the eye. All these are easily managed through effective national level programs, since in each of these cases, ready and effective solutions are available, and no new research is needed. But the rest of the blindness burden is due to corneal dystrophies, glaucoma, retinal disorders and inherited problems. In these instances, basic research and its application and translation are badly needed. Fortunately, the status of research in these areas in India is of the same level and quality as elsewhere, and the connection between basic researchers and clinicians is good and mutually productive, particularly in ophthalmology. We need to take advantage of this combination and hence the need for translational research on eye diseases.

The programme support has the following R&D projects:

FOCAL THEME 1: STEM CELL BIOLOGY AND THERAPY

Project A:  Establishment of a centre for translational research in stem cells  

FOCAL THEME 2: MOLECULAR GENETICS OF OCULAR DISEASES

Project B:  Translational research in the genetics of retinal diseases

Project C:  Molecular genetic study of retinopathy of prematurity

Project D: Identifying candidate genes associated with raised intraocular pressure in primary open angle glaucoma

Project E: Molecular phenotyping of genetic mutations leading to eye disorders – functional studies on the disorder-associated proteins

2. Establishing a “Risk assessment chip” for the eye disease age related macular degeneration

AMD is the most frequent cause of blindness in Australia and has been recognized as an increasingly major cause of blindness in India. AMD is now known to have strong genetic as well as environmental risk factors, the latter including smoking, diet, inflammatory factors and vascular parameters. Discovery of the CFH gene and other susceptibility genes have led to a renewed push to identify those gene variants as well as explore gene-environmental interactions associated with disease. This project seeks to identify variants through genome-wide association study using high density SNP and copy number microarray that would accyrately identify disease risk across different ethnic groups in Australia and India. The future outcomes would be to offer targeted treatment options to the susceptible patients thereby improving their quality of life and reducing the burden of AMD in the wider community.

3. Champalimaud Translational Centre for Eye Research

The Champalimaud Translational Centre for Eye Research has been set up at LVPEI in January 2008. The Champalimaud Foundation, based in Lisbon, Portugal, was set up on the bequest of the late Portuguese entrepreneur and philanthropist, António de Sommer Champalimaud. The Foundation has a long-term commitment to support medical research, in particular neurology, oncology and vision sciences. The Champalimaud Foundation and L V Prasad Eye Institute have agreed on the development of a vast program of translational research through the establishment of the Champalimaud Translational Centre for Eye Research or C-TRACER. Research at C-TRACER will focus on the following areas:

• Research and therapy through the utilisation of stem cells in vision-related illnesses
• Translational research utilising existing and novel clinical methodologies with the objective of furthering the knowledge of vision in ways that can be readily applied to patients
• Exchange programs involving scientists chosen by LVPEI and the Champalimaud Foundation
• Introduction of capacity building programs for developing Lusophone countries such as Mozambique, Angola, Timor and others
• Organize the Antonio Champalimaud Symposium on January 30th of every year, during which leaders in the area of ophthalmology from the world, as well as distinguished scientists from India, are invited to lecture.

4. Cultivation and characterization of human lacrimal gland cells for potential clinical application for post radiotherapy xerophthalmia

After following due procedure, lacrimal glands and conjunctival tissues were harvested from patients undergoing exenteration. The harvested tissue was explanted on denuded human amniotic membrane and uncoated tissue culture dishes. Their growth potential on both the substrates was evaluated. Initial results with three samples show that the enzyme cocktail of collagenase and hyluronidase gives better viability of cells on isolation. The lacrimal gland epithelial cells (LGEC) show better growth on denuded human amniotic membrane while on uncoated tissue culture dishes the growth of fibroblast cells is greater. We also observed mixed populations of epithelial and fibroblast cells in our cultures.

At present we are trying to optimize the culture condition to get a pure yield, growth and proliferation of lacrimal gland epithelial cells. These cells will then be characterized for the known epithelial and mesenchymal markers by confocal microscopy, FACS, IHC, RT-PCR methods. The secretory function of the LGEC will be done on the conditioned medium for the presence of IgA, lactoferrin and lysozyme by sandwich ELISA method.

5. Study of the role of vitreous liquefaction in age-related nuclear cataract

This is the Indian component of a US-India collaborative study, The US part is being done by Dr. David Beebe’s group (in particular De. Ying-Bo Shui there). The goal of this collaborative study will be to determine whether nuclear cataracts are associated with increased degeneration of the vitreous body in Indian patients, as they are in patients from the US. At the same time, this study will establish whether or not the brunescent nuclear cataracts, as found in India, have a similar association with degeneration of the vitreous body.

Degeneration of the vitreous body of the eye is a major risk factor for nuclear cataracts (Harocopos et all. 2004). These finding are consistent with the well-known association between the loss of vitreous structure during vitrectomy and nuclear cataracts, and indicates that the gel structure of the vitreous body protects the lens from nuclear cataracts. These observations, along with measurements of oxygen levels in the eye of vitrectomy patients (Holekamp et all.2005) and studies by other labs (Barbazetto et al. 2004), have led to the hypothesis that increased exposure of the lens to oxygen from the retina is the primary cause of nuclear cataracts. Therefore, the state of the vitreous body is a critical factor protecting the lens from nuclear cataract formation in older individuals.

These studies will be conducted using the essentially the same methods as in Harocopos et al. 2004, with some improvements in instrumentation. For the proposed studies, digital photographs will be taken of all of the lenses. This will permit us to grade the type and severity of cataract in India, then send the digital photos to St. Louis for confirmation by Dr. Ying-Bo Shui, a member of the Beebe group and an expert in cataract classification. The availability of photos strengthens the reliability of the proposed work. Based on our preliminary studies, we anticipate that the data can be collected and analyzed and the results submitted for publication with two years of support.

6. An advanced surface for the cell therapy of limbal epithelium for ocular surface disease: Proof of concept and clinical trials

This project aims to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells to treat blinding ocular surface diseases. The project also includes a ‘proof of concept’ clinical trial to establish the safety and efficacy of this technique in comparison to the established technique of corneal pithelial transplantation using amniotic membrane as a culture substrate. The plasma polymer coating technique allows exact control over the film thickness and has formed the basis for an established skin cell therapy in the treatment of chronic wounds and burns. By applying this technique to the cornea, we have the potential to develop a novel method for the culture and transplantation of corneal epithelium. As a part of initial standardizations and surface compatibility testing, we tried four different polymer chemistries for their suitability to promote corneal epithelial cell adhesion, proliferation and differentiation by using both explant and isolated single cell culture methods. Further investigations would help identify a suitable substrate for coating contact lens surfaces.

7. Molecular characterization of nonsporulating flamentous fungi isolated from microbial Keratitis

Microbial keratitis remains a leading cause of blindness worldwide. Among severe infective forms of keratitis, fungal keratitis is the most common in many developing countries like China, India, Ghana and Nepal. More than 105 species of fungi, classified in 56 genera, have been reported to cause mycotic keratitis.Of the different filamentous fungi isolated from mycotic keratitis there were significant number of unidentified fungi due to lack of sporulation. Accurate identification of these fungi is essential for the selection of appropriate antifungal drug for treating these infections. The aims of the study are to assess the species distribution of nonsporulating molds isolated from patients with mycotic keratitis by molecular methods and to study the treatment outcome of keratitis caused by nonsporulating molds.

8. Evaluation and Characterization of cancer stem cells in Retinoblastoma tumor

Retinoblastoma (Rb) is an intraocular tumor that grows rapidly, threatening sight and life. As in other tumors, there is increasing speculation that the Rb tumor also contains cancer-stem like cells, which could influence the prognosis and response to therapy. This study attempted to identify putative stem cells by characterizing different sub-populations of cells in retinoblastoma based on light scatter properties and differential expression of markers. Freshly isolated tumor cells obtained from 7 unfixed eye specimens were analyzed for the presence of CD44, ABCG2, CXCR4, CD133, and CD90 using fow cytometry. Dead cells were excluded by appropriate gating. RT-PCR was performed to analyze the expression of human Syntaxin1A, PRoX1, CD133 and NSE in the sorted sub-population of tumor cells. Based on light scatter properties, we observed two different sub-populations of cells in 7 samples. The small cells, assigned FSClo/SSClo (Forward scatter low/Side scatter low, ranging from 1.7% to 17.7%), were characterized as positive for CD44 and negative for CD133, CXCR4 and CD90. The large cells were designated as FSChi/SSClo (Forward scatter high/side scatter low, ranging from 2.7% to 35.1%) and characterized as positive for CD44, CD133, CXCR4 and CD90. Semi-quantitative RT-PCR analysis revealed that sorted cells of FSClo/SSClo sub-population expressed retinal progenitor cell markers PRoX1 and Syntaxin1A. In contrast FSChi/SSClo sub-population has shown low expression of these markers. We conclude that retinoblastoma, on fow cytometric analysis, reveals two distinct sub-populations expressing variability in stem cell and retinal progenitor markers. Clonal assay of the two different subpopulations and differentiation assays are under way.

9. Genetic and phenotypic comparison of primary congenital glaucoma in India and Brazil

Primary congenital glaucoma (PCG) results in an irreversible blindness in children and is largely attributed to mutations in the CYP1B1 gene. While the estimates of mutation frequencies vary widely across populations, there are subtle commonalities with respect to the prevalent mutation among different populations on a uniform haplotype background as evidenced from the mutation spectrum among PCG patients from Brazil and India. The present study would try to unravel the similarities and dissimilarities in the genetic basis of PCG among the Brazilian and Indian patients and understand the origin and migration of the mutations across these populations.

10. Central India Eye & Medical Study

The Central India Eye and Medical Diseases (CIEMS) is a large population study in a rural central Indian region (Nagpur) on 5000 individuals above 40 years of age. This study aims to understand the prevalence of various eye and other systemic diseases in this population. As part of this study, we would characterize the genetic risk factors in this population for the prevalent eye diseases based on whole genome and candidate gene screening, genotype-phenotype correlation and gene-environment interactions.

11. Genetic comparison of primary congenital glaucoma in India and Tunisia - Understanding the possible founder effects

Primary congenital glaucoma (PCG) attributed to mutations in the CYP1B1 gene affects different populations worldwide. These mutations exhibit a strong geographical clustering based on a uniform haplotype background as evidenced from the mutation spectrum among PCG patients in Morocco, Saudi Arabia and India. So far, there are no reports on the genetics of PCG in Tunisia. The present study would try to unravel the similarities and dissimilarities in the genetic basis of PCG in Tunisian and Indian patients and understand the origin and migration of common mutations across these two populations.

12. Developing a global blueprint for zero PCG: Understanding the molecular basis of PCG

In continuation to the ongoing genetics work in PCG, the resent study is based on identifying the uncharacterized genes in PCG through the model of human evolution. The remise is based on the background of the evolution and migration of gypsies from India. The cohort comprises an isolated population (gypsies) derived from an ancestral population (Indian), which was founded in the recent timescale of evolution, as a result of which it would have undergone a minimum number of genetic recombinations.

Gypsy patients exhibit 20% involvement of CYP1B1 mutations in PCG, while it is around 40% for Indian patients. The study employs a GWAS in PCG cases and controls in both the cohorts using the 1.8 million SNP Affymetrix array. We plan to derive the regions of shared haplotypes across the genomes and identify the conserved regions between these two groups, which would harbor mutations in gene(s) causing PCG.

13. Generation of disease specific inducted pleuripotent stem cells from dermal fibroblasts of patients with retinal dystrophy

Retinitis pigmentosa is a progressive genetic disorder resulting in gradual degeneration of rod and cone photoreceptor cells of the retina leading to night blindness, gradual loss of vision which later progresses to complete visual impairment and blindness. Mutations in several genes associated with phototransduction pathway, retina specific transporters, transcription factors and vitamin A metabolism are linked to this disease. Cell replacement therapy holds a great promise in treating this disease.

Pluripotent stem cells, like the embryonic stem cells, are a valuable source for cell replacement therapy. Problems of host immune response and rejection following transplantation of cells derived from embryonic stem cells, the lack of enough approved ES lines for HLA matching of diverse ethnic groups and the associated ethical concerns pose a significant obstacle to their clinical use. In addition, it is difficult to generate patient- or disease-specific ES cells, which are required for understanding the disease physiology. Recently, adult somatic cells have been reprogrammed directly to a pluripotent state similar to the ES cells by ectopic expression of transcription factors like Oct4, Sox2 and Klf4 with/without cMyc to generate induced pluripotent stem (iPS) cells (Nakagawa et al., 2008). These adult somatic cell derived pluripotent stem cells can serve as good in vitro disease models for screening new drugs, to conduct pre-clinical trials and to understand the patho-physiology of a disease. In mouse models of sickle cell anemia, the genetic defects were corrected in vitro and the genetically corrected iPS cells were differentiated to the defective cell types and were successfully used for autologous cell replacement therapy (Hanna et al., 2007).

This study aims to establish patient-specific induced pluripotent stem cells (iPSCs) using the dermal fibroblasts cultured from skin biopsies of patients with retinal dystrophy and a known genetic defect. The patient-specific iPS lines generated could be used as an in vitro model system to understand the disease physiology and to conduct in vitro proof-of-principle experimentation to explore the feasibility of gene correction and phenotype recovery for its possible use in autologous cell replacement therapy.