Page 40 - Biennial Report 2018-20 Jun 2021

commonly used antibody markers such as anti-Oct4, anti-Sox2, anti-SSEA4 and anti TRA1-60 and
the fluorescence staining was optimized. After testing the iPSCs for pluripotency, karyotype and
lineage commitment, they were found to be suitable for further analysis.
A highly specific orthogonal Cas9 protein from Francisella novicida (FnCas9) was extensively
characterized and proven to be highly efficient in HDR based genome editing as compared to
currently used Cas9 proteins. FnCas9 based genome editing was conducted in both patient
derived iPSCs and editing of the SCD mutation was validated by deep sequencing. FnCas9 based
genome editing was also performed in patient derived HSCs and currently deep sequencing is
being done to validate the correction. The main objectives of the vertical were completed
successfully.

FnCas9 specificity was determined by deep sequencing in cells and by microscale
thermophoresis in vitro. Single nucleotide level specificity was observed for FnCas9; this high
specificty abolished off-target binding and thus decreased off target effects to a minimum. This
makes FnCas9 a valuable new addition to the gene editing toolset. A FnCas9-base editor that can
perform double-strand break free DNA editing was designed and validated and is currently being
tested for generating a beneficial HPFH mutation, which can correct the symptoms of SCD.

Re-expression of fetal
hemoglobin can resuce
sickle cell defects in
affected individuals.
Therefore, efforts are
underway to reactivate
fetal hemoglobin using
gene editing approaches.
The reactivation of HbF
in immortalized
erythroid progenitor
cells and adult CD34+
hematopoietic stem progenitor cells was demonstrated from healthy donors. Thismethodology
provides a new therapeutic approach for the treatment of sickle cell disease.

GENE CORRECTION USING HUMAN iPSCs FOR HEMOPHILIA AND BETA-
THALASSEMIA

Some genetic changes can be the sole cause for rare inherited genetic disorders, while others
may determine susceptibility to several common disease conditions, in combination with genetic
and environmental factors. For instance, mutations in the genes required for blood clotting, can
result in potentially fatal bleeding disorders like hemophilia (hf9). Another gene is HBB that
codes for Hemoglobin B which is part of an essential protein that carries oxygen in our blood.
Mutations in the HBB gene can lead to beta-thalassemia, a chronic disease marked by reduced
oxygenation of blood. Currently, patients need frequent blood transfusions, which in turn put
them at high risk for infectious diseases. IGIB has been consistently studying genetic variation in

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