Hashemite University

Genetic Repair of Retinitis Pigmentosa in Patient Derived Stem Cells

- Presented by : Belal Asa’d

Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient Derived Stem Cells

Introduction

• Retinitis pigmentosa (RP)

• Inherited ( X-linked ), degenerative eye disease

• causes severe vision impairment

• progressive degeneration of the rod photoreceptor cells in the retina

• The disease caused of point mutation

• C.3070 (T) at ORF15 of Retinis Rigmentosa GTPase Regulator gene

• Stop codon stop protein synthesis • TAG - - - - - < GAG

Stop codon- - - < Glutamate

Materials and Methods• 2 technologies used in this research paper

• Genetic editing tool :CRISPR Cas-9 ** system and

• Stem cellsInduced pluripotent stem cells ** clustered regulatory-interspaced short palindromic repeats

Making stem cells• Skin-punch biopsy taken (own patient )

• Fibroblast cells taken.. Generation ability to connective tissue

• Transcription factors added

• Induced pluripotent stem cells form (iPSCs)

• But.. iPSCs still have point mutation (TAG)

Skin-punch biopsy

Stem cells typed• Pluripotent Stem Cells

- Ability to generate any type of cells- Ex: Embryonic stem cells or iPSCs- ( Embryo ) ( Adult )

• Multipotent Stem Cells

- Generate certain type of cells- Ex: Hematopoietic stem cells

Why we don’t take stem cells from embryos directly

• Ethical issues

• Risk of immuno-mediated rejection

Testing Pluripotency

• Test Pluripotency markers (Sox2)

• Testing Ability to for forming three layers

Crispr Cas-9 editing tool

CRISPR Cas9• Composed of 2 RNA guides + Cas9 endonuclease

• Transfection of iPSCs with expression vector Cas9

• Efficiency of correct cuts was 23%

• Homology-directed gene repair (HDR) completed sequence after double strand break (DSB)

• Correction percent of the mutation (G>T) of 223 cells transfected with Cas9 was 13%

Crispr Cas9 technology

• https://www.youtube.com/watch?v=ow0X8WifP08

Results

Autofluorescence imaging (A+B) + Optical coherence tomography (C+D)

Skin-punch biopsy culture and fibroblast(E) + Alkaline phosphatase stain (F) + Immunohistochemistry and fluorescence microscopy (G,H)

Injection of iPSCs into a sever combined immunodeficiency mouse forming 3 germ line layers

Crispr Cas9 and choosing gRNA

Insertion of gRNA into expression vector alongside Cas9 nuclease

PCR products analysis by 2 different polymerases show gRNA 58 is more precise in CRISPR Cas9 cleavage of target site mutation

Sanger sequencing – Dideoxynucleotide of GTPase Regulator gene in Retinis Pigmentosa patient sample (Top) and Transfected cells

with gRNA-Cas9 expression vector (Bottom)

Deep sequencing for control and Cas9 technology

Conclusion• The efficiency of cleavage of target site 3070 G>T

for CRISPR Cas9 – (g58 - gRNA) was 23% out of 293 cell line

• Correction Rate of 23% those cells due to cell replication proofreading machinery was 13% according homology-directed gene repair (HDR)

• The next step is to convert corrected iPSCs to retinal cells and transplant it in retina of the same patient

Reference• Bassuk, Alexander G., Andrew Zheng, Yao Li, Stephen H.

Tsang, and Vinit B. Mahajan. "Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells." Sci. Rep. Scientific Reports 6 (2016)