SCDAA 46th Annual National Convention

Abstract

Authors: 
Dr. Mark Dewitt-UC Berkeley, Mrs. Wendy Magis-CHORI, Dr. Stacia Wyman-UC Berkeley, Dr. Zulema Romero-UCLA, Mr. Jonathan Vu- UC Berkeley, Prof. Dario Boffelli- CHORI, Dr. Mark Walters- USCF Benioff Children’s Hospital Oakland, Dpt. Of Hematology/Oncology: Center for Inherited Blood Disorders, Dr. Donald Kohn-UCLA, Prof. Jacob Corn –UC Berkeley, Prof. David Martin- CHORI

Sickle Cell Disease (SCD) is a recessive genetic disorder caused by a single nucleotide polymorphism (SNP) in the ß-globin gene (HBB). CRISPR/Cas9 is a gene editing technology that can be used to correct the sickle mutation directly. Here we report progress towards clinical translation of a CRISPR/Cas9 gene editing approach to treat SCD. Our focus is on direct correction of the disease mutation itself. Our approach is based on electroporation of a Cas9 ribnucleoprotein (purified Cas9 protein and a synthetic protected sgRNA) with an optimized single-stranded DNA donor which programs correction of the sickle mutation by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) from sickle cell disease donors. Our approach relies on animal-free synthetic reagents, avoiding use of viral vectors. By avoiding viral HDR donors, our approach promises to reduce manufacturing costs, and ultimately improve accessibility. We obtained surplus HSPC from participants in a sickle cell disease gene therapy trial after mobilization with plerixafor, the same source of cells in our planned clinical protocol. Using an optimized gene correction protocol, we routinely achieved >30% correction of the mutation in these cells. When differentiated into erythroblasts, 70% of hemoglobin is non-sickle, of which 40% is corrected adult hemoglobin. When transplanted into immune-deficient mice, corrected cells maintain engraftment ~10-fold better than cells edited with a AAV6 viral HDR donor. Most importantly, the corrected allele is maintained in 22% of alleles in bone marrow, a substantial improvement over previous studies in SCD. In glycophorin-expressing erythroblasts in circulating blood, the corrected allele is further enriched to 30%, evidence for ineffective erythropoiesis in sickle- or indel-containing cells.
To define the off-target genotoxicity of our sickle correction protocol, we identified off-target sites using a combination of GUIDE-seq and bioinformatics. Using a pooled primer PCR approach, we find that the vast majority of these off-targets have no detectable mutagenesis. We found that a commercially-available high-fidelity Cas9 variant can reduce off-target activity 20-fold or more in HSPCs, with no loss in on-target activity. Only one single off-target site is targeted in >0.2% of the cells. Together, these studies suggest a manufacturing protocol to correct CD34+ HSPC from SCD patients that is both safe and effective in delivering a long-term clinical benefit. We have started IND-enabling studies, and anticipate filing an IND to commence a Phase I clinical trial in 2019.