Sickle cell disease (SCD) is a devastating genetic disorder that affects ~100,000 primarily African American individuals in the USA, including 5,100 in California. In SCD, a Glu to Val point mutation in the ß-globin gene renders the resultant sickle hemoglobin prone to polymerize and damage the red blood cell. The Corn lab has used CRISPR-Cas9 genome editing to develop methods to correct the sickle allele in hematopoietic stem cells (HSCs) and, together with SCD experts at Children’s Hospital Oakland, are in the process of developing proof-of-concept for a clinical trial to cure SCD via transplantation of gene-corrected autologous HSCs from patients. Their goal in this CESCG CRP is to establish the efficacy and safety of sickle correction in HSCs via targeted and unbiased sequencing. Together with the CESCG they will use next-generation sequencing to determine the extent of allele conversion in edited HSCs both in vitro and after in vivo engraftment in a mouse model. To establish the safety of editing, they will use three sequencing-based approaches. First, they will use custom amplicon-based resequencing to quantify undesired editing events at related globin genes and at sites computationally predicted as potential off-targets based on sequence similarity. Second, they will use established cancer resequencing panels to uncover low-frequency off-target events at genes known to be involved in tumorigenesis, with a focus on annotated tumor suppressors. Third, they will use unbiased capture and sequencing methods, such as the recently described GUIDE-Seq method, to uncover off-target events in the context of the entire human genome. This approach will be critical in providing key data to move editing SCD allele towards the clinic and will also provide an important precedent for establishing efficacy and safety metrics for therapeutic gene editing in HSCs.
The Corn lab has pursued two primary lines of inquiry: first, characterization of isogenic clonal cell lines bearing mutations that cause disease (hemoglobinopathies: beta thalassemia major and sickle cell disease) and second, generation of novel genotypes believed to alter globin expression based on observation of naturally occurring mutations associated with hereditary persistence of fetal hemoglobin. All cell lines were generated from an immortalized erythroblast cell line, HUDEP-2 cells, using CRISPR/Cas9, and a ssDNA HDR donor, for precise incorporation of disease mutations. These cell lines serve as a platform to explore cellular phenotypes of both healthy and diseased cells using RNAseq.
To perform RNAseq, the Corn lab engaged with the team at CESCG to prepare libraries from these cell lines, and sequence them at high throughput. They are currently working with the bioinformatics team to analyze the data from these cells. From their collaborators, they already have some preliminary RNAseq data from the panel of disease-associated cell lines, and are validating hits using CRISPR-interference and siRNA knockdown. In the coming months, the Corn lab will seek to merge and curate these datasets, which should provide a nice resource for researchers studying erythroid maturation in both healthy and diseased backgrounds. Surprisingly, curated databases of this sort do not exist yet.
Biomarkers, protocols, clustering or other supplementary files supplied by the lab
Expression Matrix (lab-generated) | Expression matrix (UCSC) | QC Metrics