Distribution of lentiviral vector integration sites in mice following therapeutic gene transfer to treat β-thalassemia

Abstract

A lentiviral vector encoding β-globin flanked by insulator elements has been used to treat β-thalassemia (β-Thal) successfully in one human subject. However, a clonal expansion was observed after integration in the HMGA2 locus, raising the question of how commonly lentiviral integration would be associated with possible insertional activation. Here, we report correcting β-Thal in a murine model using the same vector and a busulfan-conditioning regimen, allowing us to investigate efficacy and clonal evolution at 9.2 months after transplantation of bone marrow cells. The five gene-corrected recipient mice showed near normal levels of hemoglobin, reduced accumulation of reticulocytes, and normalization of spleen weights. Mapping of integration sites pretransplantation showed the expected favored integration in transcription units. The numbers of gene-corrected long-term repopulating cells deduced from the numbers of unique integrants indicated oligoclonal reconstitution. Clonal abundance was quantified using a Mu transposon-mediated method, indicating that clones with integration sites near growth-control genes were not enriched during growth. No integration sites involving HMGA2 were detected. Cells containing integration sites in genes became less common after prolonged growth, suggesting negative selection. Thus, β-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control.

Figure 1

Lentiglobin vector and experimental design. (a) Diagram of the human β-globin (β^A) lentiviral vector [Lentiglobin, (LG)]. The 3′ β-globin enhancer, the 372 base pairs (bp) IVS2 deletion, the β^A-T87Q mutation (ACA[Thr] to CAG[Gln]) and DNase I hypersensitive sites (HS) 2, HS3, and HS4 of the human β-globin locus control region (LCR) are indicated. Safety modifications including the two stop codons in the ψ+ signal, the 400 bp deletion in the U3 of the right HIV LTR, the rabbit β-globin polyA signal and the 2 × 250 bp cHS4 chromatin insulators are indicated. βp, human β-globin promoter; cPPT/flap, central polypurine tract; HIV LTR, human immunodeficiency type-1 virus long-terminal repeat; ppt, polypurine tract; RRE, Rev-responsive element; ψ+, packaging signal. (b) Treatment and transplantation protocol. Experiments were conducted with busulfan conditioning of male β-thalassemia (β-Thal) mice followed by transplant of female β-Thal bone marrow cells (4 × 10⁵ fresh, mock-transduced, or LG-transduced cells) and monitoring of peripheral blood and pathology.

Figure 2

Hematological recovery and improvement of erythroid parameters in β-thalassemia (β-Thal) recipients of Lentiglobin (LG)-transduced β-Thal bone marrow cells. Results shown are the mean ± SD for untransplanted, control β-Thal mice (n = 5), normal unmanipulated C57BL/6 mice (n = 4), and β-Thal mice transplanted with freshly harvested cells (n = 4), mock-transduced cells (n = 4), and LG-transduced cells (n = 5). Changes in red blood cell (RBC) number, hemoglobin concentration, and reticulocyte count seen at 30 days and beyond in β-Thal recipients of LG-transduced cells were highly significant in comparison with nontransplanted, or control-transplanted β-Thal mice (P < 0.001). BMT, bone marrow transplantation; RBC, red blood cells; WBC, white blood cells; WT, wild type.

Figure 3

Long-term correction of β-thalassemia (β-Thal) disease at 9.2 months post-bone marrow transplantation (BMT). (a) Spleen size and (b) spleen weight of normal C57BL/6J (WT) mice, unmanipulated β-Thal (T) and β-Thal recipients of fresh (F), mock (M), or Lentiglobin (LG)-transduced β-Thal bone marrow. The overall weight of each mouse was similar, so correction of spleen weight by body weight did not affect the conclusions (data not shown). (c) Proportion of early erythroblasts (basophilic, Ter119^high CD71^high) as compared to late erythroblasts (orthochromatophilic, Ter119^high CD71^low) from flow cytometric analysis of the bone marrow. (d) Photomicrographs of Mallory stained spleen and liver tissue sections from representative mice showing iron deposition related to β-Thal. (e) Phenotypic analysis of different cell populations in the bone marrow according to cell surface antibody staining and flow cytometry. For all histograms, results shown are the median values (red line), the 25th and 75th percentiles (bottom and top boundary of the boxes) and individual values (dots) for normal C57BL/6J (WT, n = 5), control β-Thal mice (T, n = 4), β-Thal mice transplanted with freshly harvested cells (F, n = 4), mock-transduced cells (M, n = 4), and LG-transduced cells (LG, n = 5). (f) Median copy number in the transplanted bone marrow (BM) progenitors [(colony-forming units (CFUs)] before BMT and in donor peripheral blood leukocytes (PB), total bone marrow cells (BM), and BM progenitors (CFUs) at 9.2 months post-BMT of β-Thal mice transplanted with LG-transduced cells (n = 5). Results shown are the median values (red line), the 25th and 75th percentiles (bottom and top boundary of the boxes) and individual values (dots). (g) Relationship between transgene copy number in the peripheral blood leukocytes and hemoglobin (Hb) concentration.

Figure 4

Relative proportions of integration sites in mouse bone marrow quantified using Mu-mediated recovery of integration sites. To illustrate robust recovery and quantification, the abundance of integration sites in each mouse are presented as scatter plots showing concordance between quantification methods (left column). The y axis shows the number of times an integration site was isolated with an independent transposition event catalyzed by MuA transposase in vitro. The x axis shows the number of sequence reads per integration site. The R² values indicate the correlations between the two quantification methods for each mouse. Abundant integration sites are labeled with the name of the nearest RefSeq gene. Site abundance is also displayed for each mouse as a stacked bar graph (right column) showing the relative proportions of integration sites recovered (quantified by independent Mu-mediated integration events). Sites recovered fewer than 10 times were combined into the low frequency group.

Figure 5

Distribution of unique integration sites with respect to genomic features. (a) Heat map illustrating genomic distribution of unique integration sites. Favoring or disfavoring of a genomic feature within a window around integration sites in each data set is represented as a colored tile. The color is determined by receiver operating characteristic (ROC) curve area comparing the density of the feature near experimental sites and matched random control sites. See Materials and Methods section for explanation of genomic features. The P value for the comparison with pretransplantation liquid culture, determined by a logistic regression method that respects the pairing in the data (c-logit), is overlaid on the heatmap tile (*P < 0.05; **P < 0.01; ***P < 0.001). (b) Integration in transcription units. The proportion of unique integration sites in each data set within transcription units are shown, normalized to matched random control sites (indicated by the horizontal line). Significant differences from pretransplantation (liquid culture) sites is denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 6

Analysis of the number of insulator sequences in vector long-terminal repeats (LTRs) after transduction. (a) Diagram of the 5′ and 3′-LTRs, indicating the sizes expected for amplification products with one or two insulators. Diagrams show (i) the plasmid used to produce the vector, (ii) the vector after reverse transcription and integration into the chromatin, and (iii) the vector structure after recombination between core insulator sequences. Recombination leads to the deletion of 257 bp. Primers used for PCR are indicated; R19 and F17 specifically amplify 5′ and 3′-LTR regions, respectively. F19 recognizes sequences located between the two core insulators and amplify LTRs only if two insulators are present. (b) Amplification of 5′- (left) and 3′-LTRs (right) from mouse bone marrow DNA with specific primers. In mice 31, 33.2, and 34 most of the PCR product corresponds to amplification of 5′- and 3′-LTRs with only one insulator. The smallest DNA fragments were purified an sequenced, confirming that recombination deleted 257 bp (data not shown). (c) Amplification of DNA with primers specifically amplifying intact double core insulator.