Self-inactivating gammaretroviral vectors for gene therapy of X-linked severe combined immunodeficiency

Abstract

Gene therapy for X-linked severe combined immunodeficiency (SCID-X1) has proven highly effective for long-term restoration of immunity in human subjects. However, the development of lymphoproliferative complications due to dysregulated proto-oncogene expression has underlined the necessity for developing safer vector systems. To reduce the potential for insertional mutagenesis, we have evaluated the efficacy of self-inactivating (SIN) gammaretroviral vectors in cellular and in vivo models of SCID-X1. Vectors incorporating an internal human elongation factor-1alpha regulatory element were capable of fully restoring the lymphoid differentiation potential of gammac-deficient lineage negative cells. Multilineage lymphoid reconstitution of a murine model was achieved at a similar level to that achieved by a conventional long-terminal repeat (LTR)-regulated vector used in previous clinical trials. Functional proliferative responses to mitogenic stimuli were also restored, and serum immunoglobulin levels were normalized. The reduced mutagenic potential conferred by SIN vector configurations and alternative non-LTR-based regulatory elements, together with proven efficacy in correction of cellular defects provides an important platform for development of the next phase of clinical trials for SCID-X1.

Figures

Figure 1. Self-inactivating (SIN) gammaretroviral vectors for X-linked severe combined immunodeficiency (SCID-X1).

(a) Schematic of the SRS11 series of SIN gammaretroviral vectors for SCID-X1 incorporating internal spleen focus forming virus (SFFV) and EFS promoters. (b)Long-terminal repeat (LTR)-regulated vectors used in this study for comparison. Ψ, packaging signal; MoLV, Moloney murine leukemia virus; MPSV, Myeloproliferative sarcoma virus U3; RSV, Rous sarcoma virus U3; WPRE, mutated (devoid of X-protein ORF) Woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 2. Comparison of vector performance in cell lines and wild-type (wt) primary cells.

(a) Northern blot analysis of total RNA preparations from transduced SC1 cells. The IL2RG transcript was detected by a specific probe. The expected RNA bands (unspliced versus spliced/internal transcript) are indicated by arrows on the right. Re-hybridization with probes to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed as an internal control. (b) Flow cytometry of SC1, CTLL-2 and wt lineage negative cells transduced with the indicated MFG γc and self-inactivating (SIN) vectors (SRS11 EFS γc and SRS11 SF γc). Cells were analyzed 5 days post-transduction using phycoerythrin-conjugated anti-human CD132 antibody (y-axis). Representative examples are shown. The percentage of positive cells and corresponding mean fluorescent intensity (MFI) are indicated.

Figure 3. Transduced murine Il2rg−/− lin– cells have restored T and B lymphoid differentiation potential in vitro.

(a) Murine γc–deficient lin– cells transduced with SRS11 SF γc or SRS11 EFS γc had restored B-cell differentiation potential following co-culture on OP9-eGFP stromal layers. (b) The Il2rg−/− progenitors transduced with the self-inactivating vectors also exhibited T-cell development in vitro at comparable levels to MFG γc-transduced cells or wild-type C57/Bl6 progenitors. (c) Development of CD4 and CD8 single positive cells 31 days post-transduction (control C57/Bl6 cells were analyzed 15 days post-seeding on OP9 stromal layers). In addition to CD4+/CD8+ double positive cells (upper right quadrant), mature CD4+ and CD8+ lymphocytes are indicated in upper left and lower right quadrants, respectively. eGFP, enhanced green fluorescent protein.

Figure 4. Efficient immunological reconstitution of a X-linked severe combined immunodeficiency (SCID-X1) murine model following ex vivo gene therapy using self-inactivating (SIN) gammaretroviral vectors.

(a) Equivalent high levels of transgene-positive Il2rg−/− lin– cells are observed for the SIN gammaretroviral vectors and MFG γc following two rounds of ex vivo transduction. (b) Il2rg−/−Rag2−/−c5−/− mice transplanted with SRS11 SF γc–, SRS11 EFS γc– or MFG γc–transduced γc-deficient progenitors were assessed for lymphoid reconstitution ~3 months post-transplant (see Materials and Methods). Comparable levels of CD4+ and CD8+ T cells and mature B220+IgM+ B lymphocytes in the spleens and NK1.1+ NK cells in the bone marrow were detected for the SIN vectors and long terminal repeat (LTR)-regulated vector. (c) Thymopoiesis is successfully restored in transplant recipients, with equivalent proportions of single-positive and double-positive thymocytes detected for transplant recipients and wild-type C57/Bl6 mice. (d) Il2rg−/−Rag2−/−c5−/− mice transplanted with SFFV-eGFP-transduced Il2rg−/− progenitors failed to develop T or mature B lymphocytes or NK cells in the spleen and bone marrow. (e) Proviral copy number in splenocytes from reconstituted Il2rg−/−Rag2−/−c5−/− mice was determined by quantitative polymerase chain reaction (see Materials and Methods). Equivalent low copy number was observed for SRS11 SF γc, SRS11 EFS γc and MFG γc transplant recipients indicating that immunological reconstitution was achieved with only a single proviral integration for the majority of animals. Bars represent the mean values for each group. CFU, colony forming units; eGFP, enhanced green fluorescent protein; Ig, immunoglobulin; NK cells, natural killer cells; SFFV, spleen focus forming virus.

Figure 5. Lymphocyte function is restored following ex vivo gene therapy using self-inactivating (SIN) gammaretroviral vectors.

(a) Splenocytes from transplanted Il2rg−/−Rag2−/−c5−/− or control mice were stimulated with concanavalin A (Con A) alone or in the presence of interleukin-2 (IL-2). Proliferating cells were assessed by incorporation of 3H-thymidine and expressed as a proliferation index (the ratio of the stimulated cells to unstimulated cells). Splenocytes from mice transplanted with SRS11 SF γc–, SRS11 EFS γc– or MFG γc–transduced Il2rg−/− lin– cells exhibited increased levels of proliferation as compared to untreated γc-deficient animals. The mean + SD stimulation indices for each experimental group are shown. (*P < 0.05; **P < 0.01). (b) Serum immunoglobulin (Ig) levels in SRS11 EFS γc– and MFG γc–transplanted mice and control animals were determined by enzyme-linked immunosorbent assay. Levels of IgG detected in the sera of transplanted mice were equivalent to those detected in three wild-type C57/Bl6 controls, with levels of IgG1 and IgG2a for SRS11 EFS γc mice significantly increased as compared to untreated Il2rg−/− animals. Bars represent the mean values for each group. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 6. Interleukin-2 (IL-2)-mediated signaling is restored in SRS11 EFS γc–transduced ED-7R cells.

The γc-deficient T-cell line, ED-7R, was transduced with SRS11 EFS γc using two rounds of transduction at a multiplicity of infection of 3, yielding 90% of γc+ cells as assessed by flow cytometry (data not shown). The transduced ED-7R and control cells (mock-transduced ED-7R and IL2RG-expressing ED-7R-γc cells) were stimulated with IL-2. γc-mediated signaling in response to IL-2 was partially restored in transduced cells as assessed by the presence of phosphorylated STAT5 (pSTAT5) by flow cytometry.