Session: 801. Gene Editing, Therapy and Transfer: Poster II
Hematology Disease Topics & Pathways:
Anemias, Biological, HSCs, Diseases, Therapies, Genetic Disorders, gene therapy, Technology and Procedures, Cell Lineage, gene editing, stem cells
Previously we and others identified the A>G mutation in the GATA1 binding region of intron 1 of ALAS2 in particular XLSA families and have demonstrated the key role of this site in regulating ALAS expression. Thus, we first designed a series of sgRNAs, along with single-stranded DNA oligonucleotide donors (ssODN). After co-electroporating with Cas9 mRNA into patient-derived hiPSCs, sgRNA-1 and ssODN were selected for further experiments based on optimal correction rate (43.93±3.43% via HDR). Next, using a well-established erythroid protocol, CD34+ HSPCs of control and gene-edited groups were differentiated into erythroid cells in vitro. Surprisingly, heme biosynthesis examined by benzidine staining showed that compared with the mock cells, the gene-corrected group significantly increased the frequency of benzidine-positive cells.
To examine the multilineage differentiation potential of gene-corrected CD34+ HSCPs, we performed colony-forming unit (CFU) assays to quantify various types of colonies. Compared with mock cells, gene-edited group significantly enhanced the generation of total, CFU-GM and BFU-E colonies, suggesting higher clonogenic potential. Next, gene-corrected CD34+ HSPCs were transplanted into nonobese diabetic (NOD)/Prkdcscid/IL-2Rγnull (NPG) mice to evaluate the repopulating potential. All transplanted mice displayed engraftment in multiple organs at 10-16 weeks post transplantation, and the gene-corrected cells showed greater engraftment potential than mock group. In addition, hematopoietic reconstitution analysis indicated that the gene-corrected cells maintained normal lineage distribution, while the B cell development of mock group was impaired. Moreover, gene-editing efficiency analysis of bone marrow samples 16 weeks after transplantation exhibited high editing rate (34±7.18% via HDR ), comparable to the in vitro efficiency. The specificity of the Cas9 mRNA-based gene editing system was examined using unbiased Digenome-Seq. In total, 32 potential off-target sites were identified and deeply interrogated via targeted PCR and NGS analysis of XLSA iPSCs electroporated with Cas9 mRNA and sgRNA. No off-target cleavage events were detected at these sites, suggesting a lack of detectable off-target events.
Finally, scRNA-seq of CD34+ HSPCs from healthy donor and XLSA patients revealed more HSC/LMPP and erythroid progenitor cells in older XLSA patient. Further analysis showed that cell cycle and gene expression in older HSC/LMPP cells were significantly different from that from healthy donors and younger patients. Hence, we speculated that the compensatory differentiation of HSCs caused by long-term functional red blood cell deficiency caused the abnormal expansion of HSCs, which led to the poor hematopoiesis in elderly patients.
Our study firstly uses CRISPR/Cas9 gene-editing technology to correct the disease mutation in patient’s CD34+ HSPCs and rescues ALAS2 expression and heme biosynthesis, directly confirming that this mutation is the pathogenic factor for XLSA. In addition, we dissect the transcriptional profile of CD34+ HSCPs from XLSA patients at single cell resolution for the first time, shedding light on mechanistic insights into the XLSA pathogenesis. The robust gene-correction rates and significant function rescue in patient’s CD34+ HSPCs further suggest a curable option of gene-edited HSC transplantation for the treatment of the patients with XLSA.
Disclosures: Fang: EdiGene Inc.: Current Employment. Yuan: EdiGene Inc.: Current Employment. Yang: EdiGene Inc.: Current Employment. Yu: EdiGene Inc.: Current Employment. Zhang: EdiGene Inc.: Current Employment. Shi: EdiGene Guangzhou Inc.: Current Employment. Qi: Novogene Co, Ltd: Current Employment. Wei: EdiGene Inc.: Current Employment.
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