TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

Abstract

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may usher in a new class of medicines to treat genetic diseases of the blood and immune system. With state-of-the-art methodologies, it is now possible to correct disease-causing mutations at high frequencies in HSPCs by combining ribonucleoprotein (RNP) delivery of Cas9 and chemically modified sgRNAs with homologous DNA donors via recombinant adeno-associated viral vector serotype six (AAV6). However, because of the precise nucleotide-resolution nature of gene correction, these current approaches do not allow for clonal tracking of gene targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a novel methodology that utilizes barcoded AAV6 donor template libraries, carrying either in-frame silent mutations or semi-randomized nucleotide sequences outside the coding region, to track the in vivo lineage contribution of gene targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ∼20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in immunodeficient mice. We anticipate that this methodology has the potential to be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

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

“…1F, G). The heterogeneity in the primary recipients suggests that gene-edited functional HSCs may be at low abundance, similar to the recently observed oligo-clonality of human gene-edited HSCs 20,21 . Consistent with the idea that gene editing caused some toxicity within HSCs, gene editing reduced total donor engraftment levels in competitive transplantation assays (against 1 × 10 6 whole bone marrow cells) by >30% longterm (Supplementary Fig.…”

“…In particular, the heterogeneity in engraftment chimerism points toward oligoclonality in gene editing of functional HSCs. Interestingly, this has also been recently observed in human HSPC gene editing via barcoding analysis 20,21 , suggesting this is likely to become an important area for improvement as these approaches move toward the clinic. Unlike current human HSC culture conditions, we can now stably maintain mouse HSCs ex vivo over several weeks 16,17 .…”

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

“…However, we could still confirm engraftment of edited HSCs in secondary recipient mice ( Figure S1F,G ). The heterogeneity in the primary recipients suggests that gene edited functional HSCs may be at low abundance, similar to the recently observed oligo-clonality of human gene-edited HSCs 20 . Consistent with the idea that gene editing caused some toxicity within HSCs, gene editing reduced total donor engraftment levels in competitive transplantation assays (against 1×10 6 whole bone marrow cells) by >30% long-term ( Figure S1H ), even though few differences between mock and RNP+AAV-edited HSCs could be observed in the ex vivo cell cultures ( Figure S1I-K ).…”

“…In particular, the heterogeneity in engraftment chimerism points towards oligo-clonality in gene editing of functional HSCs. Interestingly, this has also been recently observed in human HSPC gene editing via barcoding analysis 20 , suggesting this is also likely to become an important area for improvement as these approaches move towards the clinic.…”

CRISPR nuclease off-target activity and mitigation strategies

“…One major caveat of this method is that it has limited sensitivity, depends on the animal model used, and may miss low frequency events (Sato et al, 2019). Alternative in vivo approaches have developed technology to track clonality of cell-based products following ex vivo HSPC editing and transplantation through barcodes included in the HDR template (Ferrari et al, 2021;Sharma et al, 2021) or by tracking indel diversity (Magis et al, 2022). These technologies can identify clones that have expanded abnormally and hint towards genomic events that led to the oncogenic transformation.…”