The hematopoietic system of patients undergoing Hematopoietic Stem and Progenitor Cell (HSPC) Gene Therapy (GT) is fully restored when autologous engineered HSPCs are reinfused into the patient. During this process, HSPCs go through a high level of proliferation until the hematopoietic reconstitution is complete. The impact of proliferation in HSPCs on cellular fitness and safety remains an open question. Moreover, the accumulation of somatic mutations in vivo could show differences in different hematopoietic lineages depending on their susceptibility to the negative effects elicited by the DNA damage response. Furthermore, oncogene activation in human HSPCs has been shown to trigger a chronic inflammatory response leading to hematopoietic decay. Here we studied the clonality and the accumulation of somatic mutations in different hematopoietic lineages and during hematopoietic reconstitution in mice subjected to HSPC-GT. Indeed, wild type C57 mice were transplanted with bone marrow-derived lineage negative (Lin-) cells from WT mice or tumor-prone Cdkn2a-/- mice which lack p16INK4A and p19ARF proteins and thus have no barriers against proto-oncogene activation. Moreover, to evaluate if genotoxic integrations may increase the probability of acquiring somatic mutation upon oncogene activation, Lin- cells were transduced with a genotoxic LV harboring the strong retroviral enhancer/promoter Spleen Focus Forming Virus in the LTR (LV.SF.LTR) or the safer GTlike non-genotoxic LV (SIN.LV.PGK). Mice receiving WT Lin- cells treatedwith LV.SF.LTR (N= 25) or SIN.LV.PGK (N= 24) did not develop tumors, while mice transplanted with Cdkn2a/LV.SF.LTR-marked cells (N = 24) developed tumors significantly earlier compared to mock (N = 20, p < 0.0001) and mice receiving Cdkn2a/SIN.LV.PGK-treated cells (N = 23, p < 0.0001). To evaluate the clonal dynamics of hematopoietic reconstitution, vector integration sites (IS)were identified by by Sonication Mediated Integration Site (SLiM) PCR from peripheral blood, lymphoid (B and T) and myeloid cells collected every 4 weeks post transplantation. Somatic mutations were identified by analyzing the mouse genomic portion flanking each IS using VarScan2. Overall, we detected >200,000 IS, corresponding to more than 135 Mb of genomic sequence information. We introduced a new Mutation Index (MI), which normalizes the number of mutations by clones and coverage to assess mutation accumulation rates. By this approach, we found that the MI increased over time in LV.SF.LTR-treated mice and was significantly higher when compared to SIN.LV.PGK-treated mice (p < 0.001). Notably, myeloid clones exhibited a higher frequency of mutation accumulation compared to T and B cell lineages. This phenomenon was further exacerbated in Cdkn2a/LV.SF.LTR-marked cells, indicating that the absence of barriers to proto-oncogene activation and the presence of genotoxic insertions result in progressive somatic mutation accumulation and insertional mutagenesis. These results demonstrate for the first time that by combining the assessment of acquired mutations with IS analysis at the single clone level we can identify differential accumulations of somatic mutations in different hematopoietic lineages in vivo which depend on the genotoxic potential of the vector used and the ability of the genetically modified cells to sense and react to genotoxic lesions.

Acquisition of somatic mutations after hematopoietic stem cell gene therapy varies among cell lineages and is modulated by vector genotoxicity and the activity of key cellular senescence gene

Gazzo F;Masseroli M;Calabria A;
2024-01-01

Abstract

The hematopoietic system of patients undergoing Hematopoietic Stem and Progenitor Cell (HSPC) Gene Therapy (GT) is fully restored when autologous engineered HSPCs are reinfused into the patient. During this process, HSPCs go through a high level of proliferation until the hematopoietic reconstitution is complete. The impact of proliferation in HSPCs on cellular fitness and safety remains an open question. Moreover, the accumulation of somatic mutations in vivo could show differences in different hematopoietic lineages depending on their susceptibility to the negative effects elicited by the DNA damage response. Furthermore, oncogene activation in human HSPCs has been shown to trigger a chronic inflammatory response leading to hematopoietic decay. Here we studied the clonality and the accumulation of somatic mutations in different hematopoietic lineages and during hematopoietic reconstitution in mice subjected to HSPC-GT. Indeed, wild type C57 mice were transplanted with bone marrow-derived lineage negative (Lin-) cells from WT mice or tumor-prone Cdkn2a-/- mice which lack p16INK4A and p19ARF proteins and thus have no barriers against proto-oncogene activation. Moreover, to evaluate if genotoxic integrations may increase the probability of acquiring somatic mutation upon oncogene activation, Lin- cells were transduced with a genotoxic LV harboring the strong retroviral enhancer/promoter Spleen Focus Forming Virus in the LTR (LV.SF.LTR) or the safer GTlike non-genotoxic LV (SIN.LV.PGK). Mice receiving WT Lin- cells treatedwith LV.SF.LTR (N= 25) or SIN.LV.PGK (N= 24) did not develop tumors, while mice transplanted with Cdkn2a/LV.SF.LTR-marked cells (N = 24) developed tumors significantly earlier compared to mock (N = 20, p < 0.0001) and mice receiving Cdkn2a/SIN.LV.PGK-treated cells (N = 23, p < 0.0001). To evaluate the clonal dynamics of hematopoietic reconstitution, vector integration sites (IS)were identified by by Sonication Mediated Integration Site (SLiM) PCR from peripheral blood, lymphoid (B and T) and myeloid cells collected every 4 weeks post transplantation. Somatic mutations were identified by analyzing the mouse genomic portion flanking each IS using VarScan2. Overall, we detected >200,000 IS, corresponding to more than 135 Mb of genomic sequence information. We introduced a new Mutation Index (MI), which normalizes the number of mutations by clones and coverage to assess mutation accumulation rates. By this approach, we found that the MI increased over time in LV.SF.LTR-treated mice and was significantly higher when compared to SIN.LV.PGK-treated mice (p < 0.001). Notably, myeloid clones exhibited a higher frequency of mutation accumulation compared to T and B cell lineages. This phenomenon was further exacerbated in Cdkn2a/LV.SF.LTR-marked cells, indicating that the absence of barriers to proto-oncogene activation and the presence of genotoxic insertions result in progressive somatic mutation accumulation and insertional mutagenesis. These results demonstrate for the first time that by combining the assessment of acquired mutations with IS analysis at the single clone level we can identify differential accumulations of somatic mutations in different hematopoietic lineages in vivo which depend on the genotoxic potential of the vector used and the ability of the genetically modified cells to sense and react to genotoxic lesions.
2024
bioinformatics, gene-therapy
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11311/1272743
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