Page 30 - Annual report 2021-22

Annual Report 2021-22 |

Sivaprakash Ramalingam

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Sivapraksh Ramalingam works on genomic medicine to develop curative therapies using CRISPR-Cas
based editing technology to treat diseases like sickle cell disease and other hemoglobinopathies.

His lab has developed an erythroid progenitor cellular model for sickle cell disease for the
identification of fetal hemoglobin associated regulators. To achieve this, genome-editing and piggy-
back mediated recombination was used. Initially, differentiation of BEL-A (erythroid stem progenitor
cell line) was standardized and validated through flow cytometric analysis, visible change in pellet
color and enucleation through Giemsa staining. In addition, the nucleofection program and DNA
concentration were standardized. Afterwards, HBB deletion was created in BEL-A cells and validated
through GAP PCR and Sanger sequencing.

As a curative therapy for SCD, he is working on generating Hereditary persistence of fetal hemoglobin
(HPFH) mutations that will lead to reactivation of fetal hemoglobin. CRISPR-Cas is being used to
introduce the HPFH mutation in the promoter of the gamma globin gene of the human erythroid cells.
To achieve this, HUDEP-2 cells (erythroid progenitor stem cells line) were nucleofected with sgRNA
and ssODN. The double stranded break generated by sgRNA gets repaired using ssODNs with 75 bp
homology arms on either side of the break. Transfected cells were enriched by FACS, and clonal
populations were established. GFP-positive HUDEP-2 cells were sorted by FACS and distributed into
96-well plates for establishment of single cell clonal population. The clones will be further
differentiated into erythroid cells for which the standardization has already been achieved.

In collaboration with Prof. Subrata Sinha, AIIMS, New Delhi, Sivaprakash Ramalingam is also working
on developing CRSIPR-Cas based curative therapy for Gaucher’s Disease (GD) using hiPSCs. To this end,
the sgRNAs targeting the GBA mutation L444P have been designed, cloned, and validated by checking
their cleavage efficiency. A GD-IPSC line from a GD patient with a homozygous L444P mutation was
generated. Characterization of GD-iPSCs demonstrated that these pluripotent stem cells were free of
exogenous reprogramming genes and expressed pluripotent stem cell markers, exhibited a normal
karyotype and were potential of three germ layer differentiation. Transfection and single cell isolation
in the iPSC line was standardized. Genetic correction of GD-iPSC is currently underway.
In collaboration with Vision Research Foundation, Chennai, he is developing cell-penetrating peptide
based CRISPR conjugates for direct delivery into retinal tissues. CRISPR/Cas9 based approach is being
used to selectively knock down the mutant allele while retaining the normal allele thereby relieving
the toxic effects of the mutant RP proteins. To achieve this aim, two induced pluripotent stem cell
lines from patients with autosomal dominant retinitis pigmentosa mutations (RHO c.562G>A) and RP9
(c.401A>T) were procured from RIKEN cell bank, Japan and characterized for their pluripotency by
immunocytochemistry and trilineage differentiation. To knock out the mutant allele of (RHO
c.562G>A) and RP9 (c.401A>T); in silico approach was used to screen for PAM sites that were different
between the normal and the mutant alleles. Specific sgRNA target sequence was cloned into plasmid
coding for ST1 Cas9 and Sp Cas9 for (RHO c.562G>A) and RP9 (c.401A>T) mutations, respectively. As
an initial step, the specificity and efficiency of the vectors were validated in HEK293 cells using regions
containing the PAM site specific for the Cas9 vectors. In addition, RP-iPSC lines were differentiated