Clinical Scale Gene Editing for Cell and Gene Therapy Applications

Genome editing technologies have enabled the precise manipulation of DNA sequences at targeted sites to achieve therapeutic effects. Engineered endonucleases like CRISPR-Cas9 institute site-specific DNA breaks to either knock out a gene or enhance the homology directed repair (HDR) based gene correction with an intact donor template. High-fidelity editing, multiplexing capacity, and the ease of implementing the CRISPR-Cas9 system have expanded the scope of programmable genetic manipulations to include simultaneous deletions or insertions of multiple DNA sequences in a single round of mutagenesis. Gene editing has many applications in basic and biomedical research, including disease modeling. A promising cell therapy approach for hemoglobinopathies or cancer immunotherapy involves the ex vivo gene editing of autologous hematopoietic stem cells (HSCs) or T cells before administering patients. For hemoglobinopathies, gene editing tools like CRISPR-Cas9 can be used to install a double stranded break to either knock out a gene like BCL11A or enhance the knock-in with an intact donor DNA template to correct the mutation in the beta subunit of the hemoglobin gene (HBB). Cancer immunotherapy applications use CRISPR-Cas9 to knock out the TCR locus or checkpoint modulators to improve the efficacy and clinical outcomes of the living drug. Here, we describe the use of a clinically validated, regulatory compliant, scalable electroporation platform for the high efficiency, low toxicity gene editing of hCD34+ hematopoietic stem cells (HSCs), T cells, and iPSC cells at a clinical scale for preclinical evaluation and commercial production of cell therapy products for Sickle Cell Disease, TCR therapy or in disease modeling.

Learning Objectives:

1. llustrate therapeutic level of gene correction of the SCD mutation in patient derived CD34+ HSCs cells by electroporation of CRISPR-Cas9 gene editing tools. The gene edited cells with homology directed repair (HDR) engrafted and were detectable for a significant period of time in mouse xenograft models.

2. Paraphrase the optimization of the RNP mediated genome editing using CRISPR-Cas9 and a ssODN template to engineer iPSCs for treatment of diseases like DMD and Miyoshi Myopathy.

3. Describe a clinical trial where patients were infused with an autologous CRISPR-Cas9 genetically edited CD34+ HSC commercial product aimed knocking out the BCL11A enhancer, thereby reactivating the production of fetal hemoglobin to alleviate the debilitating symptoms of SCD and beta thalassemia.