Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors.

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

Immunological barriers to haematopoietic stem cell gene therapy.

Cell and gene therapies using haematopoietic stem cells (HSCs) epitomize the transformative potential of regenerative medicine. Recent clinical successes for gene therapies involving autologous HSC transplantation (HSCT) demonstrate the potential of genetic engineering in this stem cell type for curing disease. With recent advances in CRISPR gene-editing technologies, methodologies for the ex vivo expansion of HSCs and non-genotoxic conditioning protocols, the range of clinical indications for HSC-based gene therapies is expected to significantly expand. However, substantial immunological challenges need to be overcome. These include pre-existing immunity to gene-therapy reagents, immune responses to neoantigens introduced into HSCs by genetic engineering, and unique challenges associated with next-generation and off-the-shelf HSC products. By synthesizing these factors in this Review, we hope to encourage more research to address the immunological issues associated with current and next-generation HSC-based gene therapies to help realize the full potential of this field.

Development of ß-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease.

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the ß-globin gene (HBB). Ex vivo ß-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.

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 recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use 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 observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (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.

Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.

Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the ß-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of ß-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.