Maintenance of Leukocyte Telomere Length after Transplant and Chemoselection in Macaques with Polyclonal Gene Modified Cell Engraftment

Hematopoietic stem and progenitor cell (HSPC) gene therapy relies on stable therapeutic levels of gene-modified HSC engraftment. The variant methylguanine methyltransferase gene, P140K, can increase gene modified cells levels in vivo after administration of O6-benzylguanine (O6BG) and non-myeloablative bis-chloroethylnitrosurea (BCNU). However, the biological consequences of chemoselective pressure on HSPC are unknown. It is known that HSPC transplantation causes leukocyte telomere shortening, likely due to proliferative demand on engrafted blood progenitor cells required for hematopoietic reconstitution following myeloablative conditioning. Additionally, telomere shortening is associated with chromosomal instability preceding malignant evolution in blood cells. We and others demonstrate stable telomere length in pigtail and rhesus macaques following myeloablative transplantation of autologous lentivirus gene modified HSPCs. However, the selective pressure placed on P140K-modified HSC after O6BG/BCNU treatment may contribute to telomere attrition. To test this hypothesis, we performed a longitudinal assessment of telomere length on peripheral blood leukocytes using a quantitative PCR method in five pigtail macaques. Macaques received myeloablative total body irradiation (1020cGy) followed by infusion of autologous HSPC gene-modified with either a gammaretrovirus or a lentivirus encoding the P140K transgene. In all animals, O6BG/BCNU effectively increased the contribution of gene-modified cells in the blood. However, in two animals (J02370 and M02426), chemoselection was associated with a loss in total leukocyte telomere length. We sorted modified and non-modified cells in J02370 based on a fluorescent marker, and found that gene-modified leukocytes in animal J02370 displayed significantly shorter telomeres than the non-modified leukocytes. To determine the relative number of gene-modified clones contributing to this phenomenon, we investigated gene-modified clonal contribution over time in each of the five animals. Telomere shortening in animals J02370 and M02426 correlated temporally with emergence of clonal dominance in vivo, whereas animals displaying stable leukocyte telomere length maintained clonal diversity in vivo. In both J02370 and M02426, shorter total leukocyte telomere length was stably maintained with stable levels of the respective dominant clones for up to 1,700 days. Telomere shortening in these animals could be attributed to either increased cell division during clonal outgrowth, or to clonal selection of a progenitor that originally began with shorter telomeres. Importantly, our data suggest that chemoselective pressure on transduced HSPC does not impact telomere length in the setting of polyclonal hematopoiesis. While clonality may exert a negative effect on telomere length, this level of telomere attrition is not associated with malignant transformation or bone marrow failure in this animal model.