Lymphomagenesis in SCID-X1 mice following lentivirus-mediated phenotype correction independent of insertional mutagenesis and gammac overexpression

Abstract

The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in gammac-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing gammac from the phosphoglycerate kinase (PGK) and Wiscott-Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-alpha (EF1alpha) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or gammac overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety, and suggest the existence of other ill-defined risk factors for oncogenesis, including replicative stress, in gene therapy protocols targeting the hematopoietic compartment.

Figure 1

Lentiviral vector constructs used in this study. Vectors contained the promoter elements from either the 1,177 base-pair (bp) human elongation factor-1-α (EF1α), 516 bp human phosphoglycerate kinase (PGK), or 481 bp human Wiskott–Aldrich syndrome protein (WAS) genes to drive expression of the human γc cDNA. ψ, packaging and dimerization signal; GA, fragment of the HIV-1 gag gene; cPPT, central polypurine tract; RRE, Rev responsive element; RSV, Rous sarcoma virus hybrid promoter; SD/SA, splice-donor and spice-acceptor sites; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. The locations of unique and rare restriction endonuclease sites are indicated.

Figure 2

Restoration of lymphocyte populations following lentiviral vector–mediated gene transfer. (a) Splenocytes from transplant recipients were examined by flow cytometry using antibodies against murine B220, CD3, CD4, CD8, IgM, and NK1.1. (b) Thymopoiesis in transplant recipients receiving vector-treated progenitors was examined by flow cytometry using antibodies against CD3, CD4, and CD8. (c) Small intestinal samples were stained for CD3 and revealed intraepithelial lymphocyte development in wild-type mice and recipient mice following gene therapy. Bar = 100 µm.

Figure 3

Restoration of immune function following lentiviral vector–mediated gene transfer. (a) Splenocytes from transplant recipients or wild-type mice were stimulated under conditions to promote T-cell proliferation. Proliferating cells were evaluated by the incorporation of [3H] thymidine and expressed as the ratio of counts obtained for stimulated to unstimulated cells. Gray bars, conA alone; white bars, IL-2 alone; black bars, conA and IL-2. (b) Humoral immune responses, indicated by serum IgG, IgG1, and IgG2a levels, were examined in mice transplanted with vector-treated C57Bl/6 or IL2RG^−/− progenitors, and compared to γc^−/−Rag2^−/−c5^−/− mice transplanted with untransduced IL2RG^−/− cells. Histograms represent the mean value for each group (n = 4) with error bars representing the standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.0001 (Wilcoxon rank-sum test). EF1α, elongation factor-1-α OD, optical density; PGK, phosphoglycerate kinase; WAS, Wiskott–Aldrich syndrome.

Figure 4

Analysis of T-cell receptor Vβ repertoire and CDR3 spectratyping in splenocytes from mice following lentiviral vector–mediated gene transfer. (a) Splenocytes were isolated from mice between 6 and 18 months post-transplantation, and V_β repertoire analysis was performed by quantitative RT-PCR. The percentage of each V_β family is indicated with results representing the mean of triplicate values for each group (n = 3) with error bars representing the SEM. Gray bars, C57Bl/6 control group; black bars, EF1α-γc treatment group. (b) CDR3 spectratypes of TCRV_β 12-1, 13-1, 13-2, 16, and 20 families in reconstituted mice receiving either C57Bl/6 control or IL2RG^−/− progenitor cells treated with the EF1α-EGFP or EF1α-γc vectors (m233 and m26, respectively). Normal Gaussian distribution (6–11 peaks each separated by three nucleotides) was observed for TCRV_β 12-1, 13-1, 13-2, 16, and 20 in m223 and TCRV_β 12-1, 13-1, 13-2, and 16 in m26. Restricted CDR3 spectratyping of TCRV_β 16 in m21 (C57Bl/6 control progenitor cells treated with the EF1α-EGFP vector) and TCRV_β 20 in m26 (IL2RG^−/− progenitor cells treated with the EF1α-γc vector) was observed. EF1α, elongation factor-1-α TRBV, T-cell receptor V_β.

Figure 5

Phenotypic characterization of lymphomas. (a) Kaplan–Meier survival analysis at 1 year post-transplantation. The percent lymphoma-free survival was significantly higher (P = 0.0134) for mice receiving C57Bl/6 progenitors treated with the EF1α-EGFP vector (n = 19) when compared to mice receiving EF1α-γc vector-treated IL2RG^−/− cells (n = 14). (b) Malignant blasts, isolated from primary animals, were stained with antibodies against murine CD3, CD4, CD8, and B220, and analyzed by flow cytometry. (c) Hematoxylin and eosin staining revealed effacement of much of the normal splenic architecture by proliferating lymphoblastic cells (top panel) with marked hepatic infiltration by metastatic lymphoblasts (bottom panel) in mice from the EF1α-γc treatment group. Bar = 100 µm. EF1α, elongation factor-1-α.