Lentivector cryptic splicing mediates increase in CD34+ clones expressing truncated HMGA2 in human X-linked severe combined immunodeficiency

Abstract

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where ‘emergency’ life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety.

Meeting FDA Guidance recommendations for replication-competent virus and insertional oncogenesis testing

“…A recent report observed a similar phenomenon in patients with X-linked SCID treated with a lentiviral vector also containing an insulator in the LTRs, which caused premature termination of HMGA2 transcripts and transient clonal expansion. 33 Recent adverse events in a clinical trial for sickle cell anemia point out multiple factors important to consider in IO risk, including the underlying disease, the preparative regimen, and the vector insertion. 34 In this study, two individuals developed acute myeloid leukemia/myelodysplasia (MDS) but only one patient had vector in the leukemic cells.…”

Case report: Persistent shedding of a live vaccine-derived rubella virus in a young man with severe combined immunodeficiency and cutaneous granuloma

“…No specific treatment is currently indicated for rubella virus infection ( 6 , 8 ). The progression of the rubella virus infection and waning immunity, as indicated by a decline in CD4 + and CD8 + T-cell numbers in the absence of available anti-viral therapy for rubella, provided the rationale for the patient to undergo ex vivo lentiviral gene therapy at the National Institutes of Health (NCT01306019) with busulfan (6 mg/kg total) myeloid conditioning in April 2021 ( 9 , 10 ). In September 2021, approximately 5 months after gene therapy, the patient was admitted for facial rash and meningitis.…”

Multiplex HDR for disease and correction modeling of SCID by CRISPR genome editing in human HSPCs

“… 30 This resulted in successful therapy for SCID-X1 patients; however, expansion of a single clonal population was reported in one patient. 31 , 32 , 33 , 34 The main disadvantages of using γRV and LV vectors for CD34 + HSPC gene therapy remain the semi-random integration and constitutive expression of the transgene, which can lead to incomplete phenotypic correction, dysregulated hematopoiesis, toxicity, and insertional mutagenesis. 35 Thus, delivery of the transgene via a targeted genome-editing approach could prove beneficial.…”