Page 41 - Biennial Report 2018-20 Jun 2021

the Indian population for two decades, both in patient cohorts as well as the normal population,
with the overarching goal of identifying genetic markers that may enable early diagnosis, lifestyle
changes and corrective measures. In recent years, two game changing advances in biological
research can now enable researchers to not just map genetic variations, but also build tools to
correct them. First is the rapid advancement in gene editing technology that allows us to design
specific guide RNAs, which in combination with a protein can cut and replace the defective piece
of genetic material. This forms the basis of the much-celebrated CRISPR technology. Second is
the discovery of the combination of four proteins that form the core of induced pluripotent stem
cell (iPSCs) technology, which can reprogram adult cells to stem cells. Such cells continue to
replicate in the body and give rise to new cells that can in turn differentiate into different cell
lineages. At IGIB, Sivaprakash Ramalingam is supported by the DBT Ramalingaswami fellowship
to explore a program whose key steps include design and synthesis of guide RNAs to target hF9
gene sequence and the HBB gene sequence at the precise disease-causing mutation sites. These
guide RNAs, after appropriate validation in mammalian cells, could be used for correcting
hemophilia mutation in human iPSCs followed by differentiation into liver cells (hepatocytes).
The goal is in situ gene correction of HBB mutation in human iPSCs and further differentiation of
these stem cells into erythroid cells.

Eight patient donors were identified, with the help of Thalassemia and Sickle cell society (TSCS),
Hyderabad, for the beta-globin genetic correction in human iPSCs using targeted genome
engineering approach. Although the protocols are as described in the international community,
adoption of these advanced technologies in India required iPSC generation followed by several
iterations of optimization, characterization of genotype and establishment of differentiation
protocols. The human iPSCs have been now well characterized by immunocytochemistry,
quantitative real-time PCR, karyotyping and trilineage differentiation showing that these human
iPSCs are bonafide pluripotent stem cells. The beta-thalassemia mutations have been corrected
using gene-editing and the methods for differentiation of gene corrected hiPSCs into
hematopoietic stem cells have been optimized.

The know-how has been made available to the Indian scientific community through a workshop
co-organized with Indian Institute of Science and a meeting of Sickle Cell Disease and
Thalassemia clinical experts and basic research scientists from across India at IGIB. The technical
know-how is applicable to a variety of gene editing projects and is not just limited to human
diseases. For instance, IGIB scientists have lent their expertise for a collaborative programme
with SRM university on generation of inheritable, transgene-free abiotic stress (salinity and
drought) tolerant and semi dwarf Indica rice cultivars using a new plant breeding approach
funded by the Department of Biotechnology.
To achieve completely matured, capability for definitive in vivo engraftment and functional
hematopoietic stem cells from human iPSCs, efforts are underway at Sivaprakash Ramalingam’s
group to knock-in certain regulatory genes using genome engineering approach

UNDERSTANDING THE CONTRIBUTION OF INTER-INDIVIDUAL GENOMIC
DIFFERENCES IN AFFECTING CELLULAR PROTEOSTASIS

In this project from DST through the Swarnajayanti scheme, Kausik Chakraborty is trying to
elucidate the different factors that affect genotype to phenotype mapping. Genotypes guide
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