Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy

Abstract

Hematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3⁺ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.

Graphical abstract

Figure 1.

Competitive injection of SCID-X1 dogs with EF1α and PGK FV vectors. (A) Dogs R2258 and R2260 were injected with a combination of FV vectors PGK.γC.FV and EF1α.γC.FV containing the fluorophores eGFP or mCherry. (B) Kinetics of lymphocyte reconstitution (lymphocytes per microliter of peripheral blood) in R2258 and R2260. Range of lymphocyte counts in healthy dogs is shown by horizontal dashed lines. (C) Long-term analysis of gene marking in peripheral blood lymphocytes from R2258 and R2260 for the PGK and EF1α FV vectors based on fluorophore expression. Lymphocyte population was defined based on forward and side scatter. T2A, thosea asigna virus 2A self-cleaving peptide.

Figure 2.

Enhanced T-lymphocyte reconstitution with G-CSF/AMD3100 treatment before FV vector injection. (A) Schematic of experiment involving G-CSF/AMD3100 treatment before FV vector injection. (B) Flow cytometry plot of peripheral blood CD34⁺ cells in nonmobilized (H866) or mobilized (H867) newborn canines at 6 hours posttreatment. (C) Kinetics of gene marking based on fluorophore expression in circulating lymphocytes from dogs treated with different FV vectors with or without G-CSF/AMD3100 mobilization. Lymphocyte population was defined based on forward and side scatter (SSC). (D) Kinetics of lymphocyte reconstitution (lymphocytes per microliter of peripheral blood) in the same animals described in panel C. (E) Kinetics of CD3⁺ cells reconstitution (cells per microliter of peripheral blood) in the same animals described in panel C during the first 7 months posttreatment. In panels D and E, normal range of lymphocyte/CD3⁺ cell counts is shown by horizontal dashed lines. Animal R2203 only survived for 119 days posttreatment. (F) FV vector copy number measured longitudinally in peripheral blood leukocytes from unmobilized animals R2258/R2260 and mobilized animals H864/H867. BID, twice per day.

Figure 3.

Clonal diversity as determined by RIS analysis in nonmobilized and G-CSF/AMD3100-mobilized dogs before FV vector injection. (A) Clonal diversity in nonmobilized canines R2258 (left) and R2260 (right) at the indicated time points posttreatment. (B) Clonal diversity in mobilized canines H864 (left) and H867 (right) at the indicated time points posttreatment with G-CSF/AMD3100 and vector PGK.γC.FV. In all graphs, unique RISs are plotted based on the number of times the RIS was sequenced and normalized to the percentage of total RISs captured at each time point for each animal. Total number of unique RISs is shown on top of each bar. Captured RISs appearing at a frequency >1% in each sample are represented by boxes in each graph. Boxes are colored in white if they were identified at a single time point or in matching colors if they were identified in >1 time point at a frequency >1%. The gray portion of the graph depicts all RISs with a frequency <1% at each time point. Legends describing the precise chromosomal location of RISs are given in supplemental Figure 4.

Figure 4.

Thymic output in FV vector–treated animals with and without G-CSF/AMD3100 mobilization. (A) Fraction of CD45RA⁺ cells within the CD3⁺ population in peripheral blood of nonmobilized animals R2258 and R2260. (B) Fraction of CD3⁺CD45RA⁺ cells in animals H864 and H867 treated with G-CSF/AMD3100 mobilization and vector PGK.γC.FV. (C) TREC levels in peripheral blood of the same animals shown in panel A. (D) TREC levels measured in the same animals shown in panel B. In panels A and B, dashed line shows average percentage of CD45RA⁺ cells from normal dog; in panels C and D, dashed line shows TREC levels from a normal littermate control.

Figure 5.

TCR diversity as determined by TCR vector β spectratyping in mobilized FV vector–treated dogs. Rearrangement of the TCR β chain was assessed by PCR amplification of complementary DNA using 17 different primer pairs (annotated on top) at various time points posttreatment in a normal littermate control H866 (A) and in treated SCID-X1 dogs H864 and H867 (B).

Figure 6.

Validation of T-lymphocyte function in cells obtained from FV vector–treated SCID-X1 canines. (A) pSTAT3 was measured in PBMCs isolated from animals R2258 and R2260 (nonmobilized; 485 days posttreatment) or from a normal littermate control and cultured in vitro with no, low, or high levels of IL-21. pSTAT3 signal is gated from CD3⁺ cells. (B) Proliferative response to phytohemagglutinin of PBMCs isolated from the same animals and same time point shown in panel A. Cell proliferation was determined by dilution of CellTracker dye.

Figure 7.

Immunoglobulin responses in treated SCID-X1 canines. (A) Bacteriophage immunoglobulin response in serum of FV-treated SCID-X1 dogs (mobilized, green; nonmobilized, blue) and in a normal control (red) ±2 standard deviations (dashed lines). Animals were injected with a first dose of bacteriophage φX174 at 8 to 12 months post–FV vector treatment and with a second dose 6 weeks later. Primary and secondary immune responses to injection were assessed at 1, 2, and 4 weeks, compared with preinjection levels, and expressed as the rate of phage inactivation or K value (Kv; described in Methods). (B) Quantitative measurement of the 3 main classes of immunoglobulin in serum of mobilized animals at 14 and 15 months posttreatment as compared with a normal control. Standard range is based on normal dog age 1 year.