Cas9-AAV6 Gene Correction of Beta-Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice

Abstract

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a novel paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated. Here, we used a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observed a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

A Single Cell Cloning Platform for Gene Edited Functional Murine Hematopoietic Stem Cells

“…Such an approach would also be highly applicable to settings where on-target mutations are detrimental and limit the curative potential of gene correction approaches, e.g. correction of the hemoglobin sickle allele (Wilkinson et al, 2021).…”

“…The low allelic chimerism of Prkdc HDR and high abundance of Prkdc indel among myeloid cells demonstrate the challenge of ensuring that all hematopoietic cells are supplied by a genetically defined population of edited HSPCs. The potentially negative consequences of on-target indels has also recently been highlighted in other gene correction models (Wilkinson et al, 2021). This drawback inspired us to establish a single cell HSC expansion system that would allow sequence-based selection of bona fide HSCs at the clonal level.…”

Base editing of haematopoietic stem cells rescues sickle cell disease in mice

“…Genetic manipulation to induce expression of HbF does not eliminate β S , and when mediated by double-stranded DNA breaks (DSBs), carries risks associated with uncontrolled mixtures of indels (insertions Article and deletion), translocations, loss of large chromosomal segments, chromothripsis, and p53 activation [13][14][15][16][17][18] (Supplementary References). Cas9 nuclease-mediated homology-directed repair can correct HBB S but is difficult to achieve efficiently in repopulating HSCs 19,20 and also requires DSBs. Elimination of the root cause of SCD by converting the HBB S allele to a benign variant without introducing DSBs could overcome these limitations.…”

Future Prospects of Gene Therapy for Friedreich’s Ataxia

“…In this context, AAVs can remain episomal, however some studies have also shown vector integration through both non-homologous sites and homologous recombination [ 34 ]. Overall, these vectors can achieve long-term expression and are being combined with CRISPR (AAV-based CRISPR systems) to study potential treatments for β-hemoglobinopathies like sickle cell disease (ex vivo gene therapy) [ 35 ]. Another additional concern is the size of the gene that we aim to deliver.…”