Barcoding of Macaque Hematopoietic Stem and Progenitor Cells: A Robust Platform to Assess Vector Genotoxicity

Abstract

Gene therapies using integrating retrovirus vectors to modify hematopoietic stem and progenitor cells have shown great promise for the treatment of immune system and hematologic diseases. However, activation of proto-oncogenes via insertional mutagenesis has resulted in the development of leukemia. We have utilized cellular bar coding to investigate the impact of different vector designs on the clonal behavior of hematopoietic stem and progenitor cells (HSPCs) during in vivo expansion, as a quantitative surrogate assay for genotoxicity in a non-human primate model with high relevance for human biology. We transplanted two rhesus macaques with autologous CD34+ HSPCs transduced with three lentiviral vectors containing different promoters and/or enhancers of a predicted range of genotoxicities, each containing a high-diversity barcode library that uniquely tags each individual transduced HSPC. Analysis of clonal output from thousands of individual HSPCs transduced with these barcoded vectors revealed sustained clonal diversity, with no progressive dominance of clones containing any of the three vectors for up to almost 3 years post-transplantation. Our data support a low genotoxic risk for lentivirus vectors in HSPCs, even those containing strong promoters and/or enhancers. Additionally, this flexible system can be used for the testing of future vector designs.

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

“…Importantly, IS can also provide evidence of potential concerning integration patterns in tumorsuppressor genes, like PTEN 6 , TET2 7 and NF1 8 , which can be closely monitored during long-term follow-up to predict future severe adverse events. Genetic barcoding on the DNA level has been used to track the in vitro 9 and in vivo [10][11][12][13] clonal dynamics of heterogeneous mammalian cellular populations and offers several advantages over lentiviral IS tracking, although it has not been used clinically. First, the amplified region is known and nearly the same for each barcode simplifying recovery from targeted cells, as opposed to semi-random LV integrations, which require amplification of unknown sequences.…”

Advances in preclinical hematopoietic stem cell models and possible implications for improving therapeutic transplantation

“…Nonhuman primate transplantation studies offer a valuable preclinical bridge for this, due to their closer phylogenetic proximity to humans compared with mice and zebrafish. 67,68 Early work tracking the cell cycle status of baboon HSPCs indicated these cells are extremely quiescent, consistent with the murine lineage tracing studies demonstrating minimal activity of HSCs during steady-state hematopoiesis. 69 Using rhesus macaque autologous transplantation with lentiviral cellular barcoding, the Dunbar lab demonstrated that contributions from short-term HSCs and uni-lineage engrafting progenitors disappeared by 2-3 months post-transplantation, while long-term HSC contributions persisted with similar clonal dynamics beyond 3 months.…”

“…While hematopoiesis is highly conserved in vertebrate and mammalian models, a challenge remains to translate the significance of mouse and zebrafish findings into human HSCT practice. Nonhuman primate transplantation studies offer a valuable preclinical bridge for this, due to their closer phylogenetic proximity to humans compared with mice and zebrafish 67,68 . Early work tracking the cell cycle status of baboon HSPCs indicated these cells are extremely quiescent, consistent with the murine lineage tracing studies demonstrating minimal activity of HSCs during steady‐state hematopoiesis 69 .…”

“…Using rhesus macaque autologous transplantation with lentiviral cellular barcoding, the Dunbar lab demonstrated that contributions from short‐term HSCs and uni‐lineage engrafting progenitors disappeared by 2‐3 months post‐transplantation, while long‐term HSC contributions persisted with similar clonal dynamics beyond 3 months 67 . In contrast to the low number of HSCs contributing in murine transplantation, long‐term HSC contribution in macaques was clonally diverse even at 38 months post‐transplantation with no single clone predominating 68 . Interestingly, reports identified that some HSCs or multipotent progenitors have a strong skewing to produce natural killer cell lineage (CD16 + CD55 − ), lacking clonal overlap with T, B or myeloid cells 67 .…”

Clonal tracking of haematopoietic cells: insights and clinical implications

“…Beyond the scope of this review, retrieval of IS has been critical to understanding the genotoxic events linked to activation of proto‐oncogenes via integration of clinical gene therapy vectors 78,79 . Modelling the likelihood of such events via quantitative clone tracking in animal models, via detection of pre‐malignant clonal expansions, is an approach to predict genotoxicity of new vectors prior to clinical introduction 80,81 . In addition, clonal tracking can be used to understand the impact of ex vivo expansion on HSPC function or numbers, or associate engraftment with particular phenotypic classes of HSPCs 28 …”