A macaque clonal hematopoiesis model demonstrates expansion of TET2-disrupted clones and utility for testing interventions

Abstract

Individuals with age-related clonal hematopoiesis (CH) are at greater risk for hematologic malignancies and cardiovascular diseases. However, predictive preclinical animal models to recapitulate the spectrum of human CH are lacking. Through error-corrected sequencing of 56 human CH/myeloid malignancy genes we identified natural CH driver mutations in aged rhesus macaques matching genes somatically mutated in human CH, with DNMT3A mutations being the most frequent. A CH model in young adult macaques was generated via autologous transplantation of CRISPR/Cas9-mediated gene-edited hematopoietic stem and progenitor cells (HSPCs), targeting the top human CH genes with loss-of-function (LOF) mutations. Long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared to minimal expansion of clones bearing other mutations. Although the blood counts of these CH macaques were normal, their bone marrows were hypercellular and myeloid-predominant. TET2-disrupted myeloid colony-forming units isolated from these animals showed a distinct hyperinflammatory gene expression profile compared to WT. In addition, mature macrophages purified from the CH macaques showed elevated NLRP3 inflammasome activity and increased interleukin (IL)-1b and IL-6 production. The model was used to test the impact of IL-6 blockage by tocilizumab, documenting a slowing of TET2 mutated expansion, suggesting that interruption of the IL-6 axis may remove the selective advantage of mutant HSPCs. These findings provide a model for examining the pathophysiology of CH and give insights into potential therapeutic interventions.

Investigation of the Impact of Clonal Hematopoiesis on Severity and Pathophysiology of COVID-19 in Rhesus Macaques

“…Previously described RMs with spontaneous DNMT3A CH expanding post-autologous transplantation (RQ859) [19,20] or CRISPR-engineered TET2 CH (ZL39 and ZH63) [19], along with control non-CH animals receiving autologous transplantation following the same total body irradiation conditioning protocol (n=3) were inoculated with SARS-CoV-2 and monitored for 12 days (Figure 1A). All animals manifested only mild signs of clinical illness, without differences between the two groups, and no evidence of significant pulmonary inflammation on repeated chest radiographs (Figure S1).…”

“…The number in brackets in the age row gives the estimated equivalent age in humans. In control animals, absence of CH somatic mutations was verified by error-corrected ultra-deep exome sequencing of a 56 gene panel associated with myeloid neoplasms [19]. All animals receiving myeloablative total body irradiation conditioning prior to autologous transplantation.…”

“…No significant changes were observed in mutation VAFs in PB/BM granulocytes following infection. In BALF, VAFs shifted in both directions following infection (Figure S4), possibly due to changes in neutrophil to lymphocyte ratio with infection, since VAFs are usually lower in lymphoid cells in the presence of CH [19].…”

“…Changes in variant allele frequency (VAF) of CH mutations following infection were analyzed in granulocytes and cellular components of BALF via targeted deep sequencing and custom pipeline as reported [19].…”

“…We previously identified naturally occurring CH in aged RMs carrying a spectrum of mutations and an incidence similar to human CH [19]. In addition, given the lack of availability of naturally aged macaques, we created a CRISPR/Cas genetically engineered CH model in younger adult macaques that reproduces the expansion of clones with TET2 LOF and hyperinflammation [19]. In this pilot study, we utilized our macaque models to ask whether disease severity and biologic characteristics following SARS-CoV-2 inoculation was enhanced in animals with either naturally occurring CH expanding post-autologous transplantation or engineered CH mutations in the DNMT3A or TET2 genes introduced via ex vivo editing followed by autologous transplantation, in comparison to similarly transplanted and conditioned middleaged adult animals without CH mutations.…”