Lentivector cryptic splicing mediates increase in CD34+ clones expressing truncated HMGA2 in human SCID-X1

Abstract A 4–8 year follow-up of vector integration site (VIS) analysis in blood lineages from patients with X-linked severe combined immune deficiency receiving lentivector gene therapy found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of HMGA2. Some patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but with no disturbance of hematopoiesis. Molecular analysis demonstrated a cryptic splice site within the cHS4 insulator generating truncated mRNA transcripts from any transcriptionally active gene containing forward-oriented intronic insert, but with detectable effect on VIS frequency only for intron 3 HGMA2 inserts. A two base-pair change at the splice site eliminated splicing activity while retaining vector functional capability. Functional analysis of lentivectors for cryptic splicing should become routine for pre-clinical safety assessment.