All ETDs from UAB

Advisory Committee Chair

Tim M Townes

Advisory Committee Members

Casey T Weaver

Christopher A Klug

Hengbin Wang

Thomas M Ryan

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Sickle Cell Disease (SCD) is a devastating inherited disorder resulting from a single DNA base pair (bp) mutation in the sixth codon of the β-globin gene. It causes severe tissue damage that can result in strokes, splenic infarction, kidney failure, liver and lung disorders, painful crises, and other complications. Currently, the only cure for the disease is allogenic stem cell transplantation, which is only available to limited patients with histocompatible donors. In order to overcome this limitation, CRISPR/Cas enhanced gene correction in sickle patient iPS and CD34+ hematopoietic stem/progenitor cells as a therapy strategy was explored. Keratinocytes from skin biopsies of human SCD patients were high efficiently reprogrammed into induced Pluripotent Stem Cells (iPSCs) with a novel Helper-dependent adenovirus-epstein-barr virus (HDAd-EBV) hybrid vector. Up to 67.9% βA allele frequency was achieved via combined using of an adenoviral vector delivered CRISP/Cas and a 70 bp single stranded oligonucleotides (ssODN) as correction template in the sickle patient iPSCs. Whole-genome sequencing (WGS) of corrected lines originally derived from two different patients demonstrated no CRISPR/Cas off-target modification detected in 1467 potential off-target sites of both lines. In another study, in vitro assembled Cas9 RNP was nucleotroporated into the SCD iPSCs together with a 91-nt single stranded oligonucleotides (ssODN) for targeted gene correction. Sanger sequencing of 96 treated single colonies revealed 84.9% allele modification and 69.8% clone correction. Treatment of CD34+ Hematopoietic stem/progenitor cells (HSPC) purified from bone marrow of sickle patient with Cas9 RNP/ssODN resulted in 85% allele modification and 41% βS to βA allele correction. Analysis of 95 hematopoietic progenitor colonies derived from the treated CD34+ cells demonstrated that 51.6% of the progenitors and 71.4% of CFU-GEMM were corrected. Besides that, high level of βA protein was expressed in the red blood cells (RBC) differentiated from the treated CD34+ cells. These results suggest that CRISPR/Cas enhanced gene replacement in patient iPS and CD34+ hematopoietic stem/progenitor cells may provide a safe and effective therapy for sickle cell disease.

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