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.

Conflict of interest statement

The authors declare no competing interests.

© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.

Figures

Fig. 1. Chronological surveillance of gene marking and polyclonal vector insertion sites (VIS) in sorted immune cells following lentivector (LV) gene therapy in X-SCID patients (n = 8).

a Surveillance of multi-lineage gene marking in sorted immune (CD3, CD14, PMN, B, NK, and CD34+) cell lineages over time following gene therapy. b Tracking of vector integration sites at different time points in sorted immune cell lineages for each patient treated with LV-gene therapy. Bar-chart shows top 256 VIS in each patient. Low-frequency clones not shown. Source data for all patient vector integration sites and vector copy number are provided as Source Data files.

Fig. 2. In vivo enrichment of HMGA2 clones with vector insertion in sense orientation and tracking of the highly enriched HMGA2 clones in all patients following LV-gene therapy.

a Unique HMGA2 VIS containing clones are detected at higher frequency and HMGA2 VIS represent a greater proportion of all inserts detected from all patient samples compared to baseline ex vivo transduced CD34+ HSC product data sets. Shown is the frequency of detection of HMGA2 VIS calculated from cumulative VIS data for each patient including all time points and cell lineages, and then normalized as counts per million total VIS detected. Frequencies were calculated separately for both orientations of HMGA2 VIS provirus (Forward/Sense versus Reverse/Anti-Sense orientation as the HMGA2 gene). The in vivo frequency is much higher than that observed from the ex vivo transduced CD34+ HSC product for sense orientation, but not for antisense orientation. Note the Y-axis is log10 scale. b Shown is the distribution and frequency of unique Vector IS across the HMGA2 gene as detected in the ex vivo transduced CD34+ HSC product data sets (upper row) compared to the in vivo patient blood lineage derived data sets (second row). The blue or red colors, respectively, denote each unique HMGA2 VIS containing clone where provirus is in the sense orientation (blue) or antisense orientation (red) of the gene. The darker the color shading the larger the unique clone. Most insertion sites are in intron 3 of HMGA2 gene, which separates the Hmga2 protein to the N-terminal AT-hook DNA-binding domain and C-terminal acidic domain. c HMGA2 VIS clone dynamics in vivo by peripheral blood lineage in each Subject. HMGA2 VIS clone frequency was calculated as percent of all VIS at each time point in different lineages. In general, the highest frequency of HMGA2 VIS clones is seen in PMN, and CD14 cells, but also can be seen in other lineages like NK and CD19 cells. Note the different scale for the y-axis (percentage of total VIS).

Fig. 3. Characterization of HMGA2-cHS4 fusion transcripts in single-cell CD34+ clones and derived-iPSCs clones with potential synergy in multi-insert clones.

a Illustration of lentivector insertion in the 3rd intron of HMGA2 gene in the bottom of the panel. Fusion transcript was generated by HMGA2 exon1-exon2-exon3 and spliced into the cHS4 SA in the 5LTR of the vector. Splicing Donor (SD) of HMGA2 exon3 and splicing acceptor (SA) of cHS4 sequences are shown on the top. PCR primers used to amplify the fusion transcript from P6 CD34 cells are located on HMGA2 exon3 and vector LTR. Amplified fusion transcript from RTPCR is shown in the right. b Quantification of HMGA2 transcript in different blood lineages in P6 and the 5′/3′ ratio, both determined by ddPCR as described in methods. HMGA2 is expressed in healthy donor (HD) CD34 cells, P6 CD34 cells, as well as P6 NK cells. It is not detected (ND) in P6 PMN, CD14, CD3, CD19 cells. The elevated 5′/3′ ratio suggest accumulation of truncated transcript relative to full-length transcript. c P6 Clonality at 30 m based on VISA. d Single-cell colony assay identified clones with multicopy transgenes. e Karyotype in iPSCs derived from P6 peripheral blood CD34+ cells. fHMGA2-10+ copy clones are the most abundant clones in peripheral CD34+ cells in PT6 and this clone is significantly enriched in iPSC cells derived from CD34+ cells (*p = 0.0002, Fisher’s exact test). Source data are provided as a Source Data file.

Fig. 4. Cryptic splice acceptor in cHS4 causes transcription termination and aberrant fusion transcripts when inserted in same orientation of almost all target genes confirmed in iPS clones.

No fusion transcript is observed for proviral integration with reverse orientation as the gene. Four iPSC clones were analyzed by RNAseq. Gene labels with the same color denote LV-insertion in the same iPSC clone. a Almost all LV insertions that are in the same orientation as the insert gene generated fusion transcripts of the upstream exons or even upstream introns into cHS4 in the LV, except for CTSS (not expressed in iPSCs, and therefore not possible to detect transcripts). Red arrows denote LV-insertion site in the gene in the same orientation. Fusion transcript sequences were identified by RNAseq and labeled above the junction. Black text indicates the sequences from upstream exons/introns and red text indicates the sequences derived from cHS4. b LV insertions in the reverse orientation of target gene or outside the gene do not produce fusion transcripts. Blue arrows indicate LV integration site and orientation relative to the gene. Insertion in TALDO1 is the only exonic insertion.

Fig. 5. Dominant termination of insert alleles due to a cryptic splice acceptor in the cHS4 region of the vector.

a Quantification of exon-cHS4 gene trap splicing and normal exon-exon splicing of target genes in single-cell iPS clones. RNAseq was perform for the iPS clones. Read counts across the exon-cHS4 splicing junction and normal exon-exon junction are reported as fraction of the total reads to measure the frequency of each splicing event. b Figure depicting the consensus sequences for splice donor, branch and acceptor sites that corresponds to the lentivector cHS4 sequence. c Vector sequence indicating branch site and cryptic splice sites. Single base-pair changes from A to T were introduced at #1 and #2 to modify the vector.

Fig. 6. Modified vector with removal of cryptic SA corrects X-SCID CD34+ HSCs.

Functional restoration of X-SCID CD34+ HSCs transduced by modified lentivector. a Stages of T-cell differentiation using the Artificial thymic organoid. b Percentages of in vitro CD34-derived T-cell progenitors. c Percentages of γ+ cells (by flow cytometry) following in vitro CD34+ differentiation into T cells. d Percentages of γ+ NK cells (by flow cytometry) following in vitro differentiation. e Phospho-STAT5 signaling in in vitro-differentiated T cells derived from transduced X-SCID CD34+ HSCs. f Vector copy number determined by ddPCR in HSCs transduced by clinical lentivector (LV) or the modified vector (LV-modified). For all graphs, data are showed as mean ± standard deviation (SD); n = 2 independent experiments. Source data are provided as a Source Data file.