Aberrant Clonal Hematopoiesis of the Erythroid and Myeloid Lineages in a Lentivirally Barcoded Rhesus Macaque

Lentiviral vectors have been used for the delivery of genetic material into hematopoietic stem and progenitor cells (HSPC) in over 100 patients enrolled in clinical trials worldwide. Lentiviral vectors, in contrast to γ-retroviral vectors, have not been associated with insertion site-associated malignant clonal expansions, and thus have been considered safer for use in HSC gene therapies. Here, however, we present a case of abberant, markedly dysplastic clonal hematopoiesis impacting the erythroid, myeloid and megakaryocytic lineages in a rhesus macaque transplanted with lentivirally barcoded hematopoietic stem and progenitor cells.

Autologous CD34+ HPSC from macaque ZL34 were transduced with a highly diverse lentiviral barcoded library and reinfused following total body irradiation. The lentiviral vector contained a copGFP reporter gene and a murine stem-cell virus (MSCV) promoter. Transduction parameters (MOI:25) and cell dose (5.4 * 10⁶ CD34+ cells/kg, 27.1% GFP+ infusate) were similar to those of 14 other macaques transplanted with the identical or similar barcoded lentiviral vectors that manifested highly polyclonal (thousands clones/animal) and stable hematopoiesis following transplantation (Koelle, Blood 2017; Wu Cell Stem Cell, 2014).

ZL34 initially engrafted with normal kinetics and blood count recovery. However, beginning 4-5 months (m) post-transplant, increased numbers of eosinophils were noted in the blood, and platelet counts decreased from the normal range to less than 30,000/uL. By 7m, strikingly elevated numbers of nucleated red blood cells (nRBCs) appeared in the blood, persisting at levels of up to 6000/uL up to the present, 16m post-transplant. Fetal hemoglobin levels were markedly increased to >76% as determined by HPLC. The bone marrow was hypercellular and markedly dysplastic, with uninuclear/small megakaryoctyes, erythroid predominance, and a left-shift in all lineages but no increased blasts. Karyotype was normal.

Barcode retrieval from purified blood myeloid, T, B, NK and nRB cell populations via low cycle PCR and Illumina sequencing revealed marked clonal dominance of 7 barcodes in the nRBC lineage and in the dysplastic myeloid lineages beginning at 5 months post-transplant. All 7 barcodes were confirmed to be present in the abnormal clone by CFU assays. High-throughput insertion site retrieval followed by insertion-specific PCR assay matched 6 barcodes to insertions, uncovered two additional mutated barcodes in the dominant clone not previously retrieved, and thus so far confirm 8-9 insertions in the dominant nRBC/myeloid clone. These insertions are adjacent to or within several potential genes of interest given the phenotype, including KITLG (SCF) and PLAG1. Bulk and single cell RNA-seq studies are being performed to uncover putative insertion-mediated upregulation or downregulation of genes near the 8 dominant integration sites.

In conjunction with our previous barcoding studies and unpublished erythropoiesis studies, these results suggest that lentiviral insertion(s) into an HSPC resulted in clonal expansion, markedly abnormal myeloid and erythroid proliferation and differentiation, and a profoundly abnormal myeloproliferative/myelodysplastic phenotype. As this is possibly the first clear case of a lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal HSPC, these results are concerning, and suggest that other methods such as targeted gene correction are important to pursue for gene therapy of bone marrow diseases.