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Research & Initiatives

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Gene editing to treat hemoglobinopathies

FnCas9 based gene editing is being developed for potential treatment of Sickle cell anemia and thalassemia.

CRISPR-Cas9 based Gene Therapy

Genome engineering through CRISPR-Cas9 is a revolutionary approach with diverse applications ranging from gene discovery to crop improvement, exploration of disease pathophysiology, development of disease models, identification of drug targets, etc. It has also been applied as a therapeutic intervention for treating various genetic diseases. Correction of a faulty gene or introduction of a desirable mutation has been shown to reverse genetic disease-associated symptoms for currently incurable and complicated pathophysiologies.

Sickle Cell Disease is monogenic in nature with a well-characterized point mutation in the HBB gene. It has one of the highest incidence rates in the Indian population. especially in low socio-economic status communities. The current treatment approaches are restricted to the use of Hydroxyurea, blood transfusions, and eventually, stem cell transplants. A crucial way to tackle this growing number of patients is the identification of carriers and counseling of patients besides finding an effective, robust, and cheap gene editing method. In this direction, our lab is working on developing base editors and CRISPR-Cas-based treatment options for SCD and other hemoglobinopathies like thalassemia. Our lab focuses on engineering highly specific and effective CRISPR-Cas9-based genome editing tools, mainly with the use of enhanced FnCas9 and base editing for developing curative therapies for Sickle Cell Disease, Duchenne Muscular Dystrophy, FENIB, etc. The lab is also exploring if the enhanced FnCas9 versions exhibit higher HDR efficiency for the same system 

Cerebral Organoids for Disease Modelling

The RNA Biology Lab cultures pluripotent stem cells to develop cerebral organoids (or ‘mini brains’). These 'organs on a dish' recapitulate the developmental plasticity of an actual mammalian brain and can be used as a great model for studying development, modeling disease pathophysiology, and discovering novel drug molecules. Cerebral organoids have a lot of potential in personalized medicine and therapy when developed from patient-specific induced pluripotent stem cells (iPSCs). Along with the labs of Souvik Maiti and Beena Pillai, we are testing small molecules involved in the growth and proliferation of mouse organoids.

 

The developed models are currently being used to understand the disease pathogenesis of Megalencephalic Leukoencephalopathy (MLC) and Spinocerebellar Ataxia (SCA) Type 17 to create disease models. These models would better simulate the disease characteristics in patients and thereafter be applied for CRISPR-Cas-based gene editing for the potential treatment of these diseases. We are currently focusing on neuronal migration inside the brain that occurs at the time of development. through assembloids (fused ventral and dorsal forebrain organoids). After attaining their position, neurons form circuits by interconnecting with each other and start acting as the basic building blocks of any brain activity that executes behavior. Any issues in circuitry could result in malformed behaviors as seen in diseases like Autism, Schizophrenia, and Epilepsy.  Another exciting area of research in the lab is to study the role of lncRNAs and their regulation during early neuronal development using cortical spheroids. 

CRISPR-Cas Based Diagnostics

Researchers at the RNA Biology Lab have developed diagnostic strategies via the CRISPR-Cas system. The high specificity and sensitivity of the system make it a valuable tool for diagnostics, particularly for identifying pathogenic or mutational signatures. FnCas9 (Cas9 isolated from Francisella novicida) due to its extremely high base mismatch sensitivity, has been adapted in the lab for the development of the FnCas9 Editor Linked Uniform Detection Assay (FELUDA). it acts as a point-of-care diagnostic kit for the detection of SARS-CoV2 variants during the COVID-19 pandemic. The kit can also successfully detect HBB variants in SCD patients and

 

The endonuclease FnCas9 is guided by a gRNA that can recognize the target genome. Complementarity leads to the binding of the ribonucleoprotein complex to the region of interest. The output can be read through multiple signal detection platforms and thus can be used for rapid diagnosis of both genetic as well as pathogenic signatures. For instrument-free visual detection, FELUDA has been adapted to be performed on lateral flow strips that capture this complex and result in a clear test band, called Rapid variant Assay (RAY). For this, FAM-labeled RNP complex (biotinylated amplicon DNA-FAM labeled sgRNA bound FnCas9) and biotinylated amplification products have been used. The complex could be visualized as it gets captured by streptavidin present on the strip test line. This method has been applied to developing a rapid, cost-effective, and instrument-free paper strip test for COVID-19. Currently, this technology has finished licensing, and approvals and is available commercially. Together with the lab of Souvik Maiti, we plan to modify the diagnostic strategy so that it can distinguish carriers from homozygotes for various genetic diseases. A web-based tool, JATAYU (Junction for Analysis and Target Design for Your FELUDA assay) has been created for the design of sgRNA and primers using FELUDA making it easily adaptable for the detection of many mutational variants. 

Read more about FELUDA here:

https://www.tata.com/newsroom/covid19/covid-19-feluda-testing-digital-solutions-tata-sons

https://www.bbc.com/news/world-asia-india-54338864

https://www.cnn.com/2020/10/05/india/india-covid-19-hour-tests-approved-intl/index.html

RNA Signatures in Cell-Fate Decisions

Embryonic Stem Cells (ESCs) exhibit the nature of pluripotency and developmental plasticity. Originally, from the inner cell mass of a human blastocyst, they have the potential to differentiate into every other cell or tissue type be it a neuron or a hepatocyte. By providing appropriate conditions in vitro, they can be made to proliferate indefinitely. This property can be manipulated for various therapeutic applications such as disease modeling and personalized medicine via lineage commitment. An embryonic stem cell can be subjected to a specific lineage by tweaking associated controlling factors.

 

In our lab, we have extensively studied such environmental cues and pluripotency regulators, particularly the splicing factor TOBF1. The overexpression or downregulation of this RNA-binding protein has been shown to interfere with the maintenance of embryonic stem cell identity. Our recent studies on TOBF1 chromatin occupancy, associated OCT-SOX binding motifs, and transcripts that undergo alternative splicing upon its disruption have helped unmask their local nuclear territories. In recent years, owing to the development of better strategies to identify and study them, lncRNAs have been implicated in a variety of different cellular pathways including disease progression. We are interested in studying functional lncRNAs that are implicated in certain types of cancer in the Indian population and developing strategies to utilize them as molecular biomarkers for early diagnosis and better prognostics. The long-non-coding RNA Panct1 interacts with TOBF1 and localizes to OCT-SOX motifs in a specific cell-cycle regulated manner and helps maintain the identity of embryonic stem cells in mice. The associated mechanistic pathways are also being explored with regard to various genetic diseases.

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