Page 39 - Biennial Report 2018-20 Jun 2021

CSIR SICKLE CELL ANEMIA MISSION

Human red blood cells are abundant cells that are dedicated to carrying oxygen to tissues, since
they contain hemoglobin, which is the oxygen carrying protein. While normal RBCs are disc
shaped, inherited changes in the sequence of the hemoglobin protein resulting in abnormal
aggregation can cause formation of sickle shaped cells. The oxygen carrying capacity of these
sickle cells is severely compromised. Sickle Cell Disease manifests in individuals who inherit two
defective copies of the gene, and suffer from chronic anaemia, swelling in hands and feet
accompanied with pain. The sickled RBCs are not easily infected by the malarial parasite, thus,
in regions where malaria is common, the sickle cell form of the gene is selected. In many parts
of India, especially in Madhya Pradesh, Chhattisgarh and Orissa, many tribal communities have
a high percentage of people with sickle cell anaemia. Under the CSIR mission mode project,
different approaches from small molecule mediated drug discovery to cell-based therapies are
being tried. At IGIB, a team of scientists including Souvik Maiti, Debojyoti Chakraborty and
Sivaprakash Ramalingam are trying to develop gene editing based methods for detection and
correction of the disease carrying beta-globin gene. The broad objectives under the genome
editing and stem cell research approach for the treatment of sickle cell anaemia (SCA, also called
as sickle cell disease or SCD) include gene editing, generation of patient specific hiPSCs followed
by design of autologous, personalized cell therapy products, using CRISPR/Cas9-directed
correction of sickle cell mutation. The correction of the mutation would be carried out in the
hematopoietic stem cells (HSCs) of sickle cell anemia patients. Functional validation of
genetically edited HSCs using ex vivo erythroid differentiation and transplantation would
ultimately pave the way for development of cell based therapy for sickle cell anaemia. Patient
identification and collection of samples is being done through a collaboration with All India
Institute of Medical Sciences (AIIMS), New Delhi. Patients who are homozygous for SCA are being
recruited; PBMCs from thesedonors will be used for foriPSC based gene correction. Five Indian
sickle cell anemia patient-derived iPSCs were generated, characterized and banked.

For development of gene editing tools, CRISPR based gene editing strategies were optimized at
three levels: DNA, RNA and protein in cultured cells. In-house synthesis of Cas9
Ribonucleoprotein components was standardized and shared with CSIR-IICB for hematopoietic
gene correction. sgRNAs for targeting the Hbb locus where genome editing will be performed
were synthesized and validated in vitro. Novel genome editing components (using CRISPR Cas9
protein/constructs) for SCD specific targeting/correction were developed, including
sgRNAs/donor template that have not been reported in literature.

Next, iPSC generation and validation were carried out. Transfection rates in pluripotent mouse
embryonic stem cells were standardized between lipofection and electroporation with the latter
working optimally for the delivery of RNPs. Currently, up to 8% genome editing by HDR was
achieved after optimization. Culture conditions of human iPSCs were standardized; freeze- thaw
cycles and differentiation into neural germ layer were optimized to ensure maintenance of
pluripotent character. Healthy donor blood was successfully reprogrammed into iPSCs through
a cocktail of Sendai virus based reprogramming factors in 15 days. A large number of colonies
per transduction were obtained suggesting a robust reprogramming efficiency. iPSC colonies
stained positive for TRA-1, a marker for pluripotency and are currently being stained and assayed
for the expression of a wide range of pluripotency markers. The cells were fixed and stained with

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