Repetitive Busulfan Administration After Hematopoietic Stem Cell Gene Therapy Associated with a Dominant HDAC7 Clone in a Nonhuman Primate

Abstract

The risk of genotoxicity of retroviral vector-delivered gene therapy targeting hematopoietic stem cells (HSCs) has been highlighted by the development of clonal dominance and malignancies in human and animal gene therapy trials. Large-animal models have proven invaluable to test the safety of retroviral vectors, but the detection of clonal dominance may require years of follow-up. We hypothesized that hematopoietic stress may accelerate the proliferation and therefore the detection of abnormal clones in these models. We administered four monthly busulfan (Bu) infusions to induce hematopoietic stress in a healthy rhesus macaque previously transplanted with CD34 þ cells transduced with retroviral vectors carrying a simple marker gene. Busulfan administration resulted in significant cytopenias with each cycle, and prolonged pancytopenia after the final cycle with eventual recovery. Before busulfan treatment there was highly polyclonal marking in all lineages. After Bu administration clonal diversity was markedly decreased in all lineages. Unexpectedly, we found no evidence of selection of the MDS1=EVI1 clones present before Bu administration, but a clone with a vector integration in intron 1 of the histone deacetylase-7 (HDAC7) gene became dominant in granulocytes over time after Bu administration. The overall marking level in the animal was increased significantly after Bu treatment and coincident with expansion of the HDAC7 clone, suggesting an in vivo advantage for this clone under stress. HDAC7 expression was upregulated in marrow progenitors containing the vector. Almost 5 years after Bu administration, the animal developed progressive cytopenias, and at autopsy the marrow showed complete lack of neutrophil or platelet maturation, with a new population of approximately 20% undifferentiated blasts. These data suggest that chemotherapeutic stress may accelerate vector-related clonal dominance, even in the absence of drug resistance genes expressed by the vector. This model may both accelerate the detection of abnormal clones to facilitate analysis of genotoxicity for human gene therapy, and help assess the safety of administering myelotoxic chemotherapeutic agents in patients previously engrafted with vector-containing cells.

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

“… 20 , 37 NHPs transplated with MLV-transduced CD34+ cells have developed clonal dominance proceeding to overt MDS and/or leukemia due to insertional mutagenesis. 21 , 22 , 43 Although overt leukemia with RV transduction of HSPCs in large animal models has been an infrequent event, 44 most studies involved follow-ups shorter than relevant for clinical trials, relatively low transduction efficiency (median of 5% gene marking), and did not clearly provide data demonstrating whether overt malignancy was preceded in time by a long period of detectable clonal expansion. Our group previously demonstrated effective LV gene transfer to repopulating NHP HSPCs and long-term polyclonal hematopoiesis, without evidence of marked clonal expansion, even utilizing a vector with an extremely strong internal viral promoter/enhancer.…”

“…Sensitivity might be enhanced or clonal expansions accelerated by the application of proliferative stress post-transplant, for instance, with busulfan, which we have previously shown can induce clonal dominance. 22 …”

“… 19 , 20 While NHP models are expensive and technically demanding, they can be used to study the behavior of thousands of vector insertion sites in a small cohort of animals over time. However, development of tumors in NHPs is rare and requires long-term follow-up; 21 , 22 thus, an approach to monitor genotoxic clonal expansion prior to actual malignant transformation would be of utility.…”

Hematopoietic Stem Cell Gene Therapy: Assessing the Relevance of Preclinical Models

“…We and others then rapidly completed surveys of large numbers of insertions in monkeys and dogs, providing the first evidence in primary cells for a number of very important principles, including a pattern of integrations favoring expressed genes with both MLV- and lentivirus-based vectors, and transcription start sites for MLV-based vectors, 283 and marked over-representation of integrations in specific proto-oncogenes such as EVI1, MDS1, HDAC7 and others, in a manner suggesting in vivo selection. 282,288 The identification of EVI1/MDS1 as a particularly worrisome locus was prescient, as the CGD trial adverse events demonstrated a few years later. 246 Novel vectors were reported as having much less concerning integration patterns in large animal models, including human foamy virus 289 and avian leucosis sarcoma virus.…”

Gene Therapy of Hematopoietic and Immune Systems: Current State and Perspectives

“…Negative selection strategies using in particular alkylating drugs place a significant stress upon hematopoietic system. However, as demonstrated by Xie et al, 2010, repetitive hematopoietic stress by busulfan administration in a nonhuman primate may rapidly lead to reduction of polyclonality and eventually to cytopenia. In addition, potential long term mutagenic effects of alkylating agents are largely unknown, thus adding more uncertainty as to correct assessment of risks and benefits of this strategy.…”