Safety Of a Gamma Globin Expressing Lentivirus Vector In a Non-Human Primate Model For Gene Therapy Of Sickle Cell Disease

Strategies for human gene therapy trials targeting hematopoietic stem cells (HSCs) are complicated by studies in murine models due to differences in stem cell behavior, short life-span and limited HSCs that could be transduced and transplanted when studying safety of viral vectors. Recent reports on adverse genotoxic events with integrating viral vectors in clinical trials utilizing autologous gene corrected HSCs underscores the need for safer gene transfer vectors. Non-human primates are relevant models due to similarities in the behavior of hematopoietic stem/progenitor cells, global gene expression profile, ability to assess long-term engraftment of transduced cells and safety of gene-modified HSCs, and thus could relatively accurately predict risk of vector genotoxicity. As a preclinical step towards globin gene therapy for hemoglobinopathies, we used pigtailed macaque HSC transplantation (HSCT) model to ascertain long-term safety and stable transgene expression from sGbG, a lentiviral vector (LV) encoding human γ-globin coding sequences from a β-globin promoter and locus control region (LCR). We observed upregulation of endogenous macaque fetal hemoglobin post-HSCT, which decreased to minimal levels by two years post-HSCT, a well-documented phenomenon following HSCT in humans. However, fetal hemoglobin (HbF) (comprised of macaque α and human γ-globin) expression remained steady at 12-15% even after 700 days post-HSCT. At 2.5 years post-HSCT, the HbF expression in a macaque transplanted with HSCs gene-modified with sGbG was stable in the range of 13% vs. 0.1% for control macaque; the average vector copy ranged between 0.13 and 0.28 with stable gene marking during the analysis period. In order to evaluate the LV integration site clonal population in sGbG transduced macaque repopulating cells, modified genome sequencing PCR was performed on genomic DNA from white blood cells and PCR products were sequenced. The junction sequences were mapped to the rhesus macaque genome assembly. A total of 177 unique vector insertions were retrieved at 6 months post-HSCT (early) and 102 vector insertions at 2.5 years (late) post-HSCT respectively. The relative distribution of vector insertions into chromosomes revealed a slight over-representation into Chromosome 16, both at early and late time points. Analysis of distribution of LV integrations of with respect to transcription start sites (TSS) revealed no insertions within the 2.5kb region of TSS. The frequency of insertions was concentrated near the 10-50kb window of TSS both upstream (18.6%) and downstream (15.6%) respectively. Interestingly, among the retrieved insertion sites, only 10% (17 insertions) were common at both time points, while 90% of insertions were unique at each time point, suggesting clonal fluctuations, with multiple HSC clones contributing to hematopoiesis at an early time point, and unique, HSC clones emerged at a later time point. Comparison of the top ten most frequently detected insertion sites at both time points revealed one insertion at Chromosome 16 mapping to an intron of KIAA0195 (an uncharacterized protein expressed ubiquitously), retrieved at both time points contributed to 3.27% and 9.23% of gene modified cells at early and late time points, respectively. No insertions were near MDS/EVI1, PRDM16 or HMGA2 loci. Other oncogenes and cancer associated genes were in the vicinity of some integrants; however, there was no significant clustering of insertions in gene regions. To assess the effect of insertions on flanking gene expression or putative cancer associated genes, we performed mRNAseq on whole blood RNA from sGbG macaque and two control macaques. A comparative analysis of transcript levels of >30,000 genes revealed no difference in global gene expression profile, gene insertions and genes within 300kb region of the LV insertion sites. Importantly, transcript levels of the most abundant clone observed (KIAA0195, Chr16: 70791901) and flanking genes, the tRNA splicing endonuclease subunit SEN54 and CASK interacting protein 2 differed from two control macaques analyzed by <2 fold. In summary, long-term follow-up data from a macaque that received cells gene-modified with a human γ-globin LV reveal polyclonal reconstitution of transduced cells, HSC clonal fluctuation, and a normal transcriptional profile, suggesting low risk of genotoxicity from this vector.

Arumugam P, Burtner C: Equal Contribution