Foamy viral vector integration sites in SCID-repopulating cells after MGMTP140K-mediated in vivo selection

Abstract

Foamy virus (FV) vectors are promising for hematopoietic stem cell (HSC) gene therapy but preclinical data on the clonal composition of FV vector transduced human repopulating cells is needed. Human CD34+ human cord blood cells were transduced with an FV vector encoding a methylguanine methyltransferase (MGMT)P140K transgene, transplanted into immunodeficient NOD/SCID IL2Rγnull (NSG) mice, and selected in vivo for gene-modified cells. The retroviral insertion site (RIS) profile of repopulating clones was examined using modified genomic sequencing PCR (MGS-PCR). We observed polyclonal repopulation with no evidence of clonal dominance even with the use of a strong internal spleen focus forming virus (SFFV) promoter known to be genotoxic. Our data supports the use of FV vectors with MGMTP140K for HSC gene therapy, but also suggests additional safety features should be developed and evaluated.

Insulated Foamy Viral Vectors

“…We also did not identify clonal dominance within any of the samples, consistent with FV vector-transduced samples being highly polyclonal with and without an insulator. 41,66 Despite these similarities, a significant reduction in the number of observed integration sites within hotspots was observed when FV vectors were insulated. The effect is seen as early as 5 days after vector exposure and increases over time as indicated by a significant increase in the number of observed integrations within hotspots in uninsulated FV vector-transduced samples (Fig.…”

“…[41][42][43] The GV vector CL-SGN was generated from pCAG-GFP, a gift from F. Gage (plasmid no. 16664; Addgene, Cambridge, MA), 44 where an SFFV EGFP neomycin cassette was cloned in between the EcoRI sites, using standard molecular biology techniques.…”

“…PERL scripts querying oncogene databases were used to assess integrations within and near known proto-oncogenes as previously described. 20,41 Retroviral integration site (RIS) data sets were divided into a minimum of three nonoverlapping randomly selected matched size data sets of 1588 unique RIS for hotspot analysis.…”

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy

“…23,24 These properties contribute to their safety as established in the canine model 21,25,26 and in the murine xenotransplantation model. 20,[27][28][29] We previously demonstrated the feasibility of in vivo gene therapy in canine SCID-X1 with IV injection of FV vector expressing human codon optimized gC driven by the short elongation factor-1 a promoter (EF1a; EF1a.gC.FV). 30 Successful lymphocyte expansion was reported in these animals, but clonal diversity and T-cell receptor (TCR) repertoire were low, which prompted further optimization of our method.…”

INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions

“…We have published statistical tools for analysis of such data and applied these tools to track several gene therapy trials 20, 21, 25, 26, 34, 35, 36, 37, 38, 39. Other groups have also used related methods 6, 8, 24, 40, 41, 42, 43, 44…”