Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer - PubMed (original) (raw)

Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer

Gustavo Mostoslavsky et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Artemis gene mutations are responsible for the development of a severe combined immunodeficiency [radiation-sensitive (RS) SCID] characterized by a severe B and T cell deficiency and a normal natural killer cell population. To establish the feasibility of a gene therapy approach to the treatment of RS-SCID, we generated a series of lentiviral vectors expressing human Artemis from different promoters and used them to transduce highly purified hematopoietic stem cells (HSCs) from Artemis knockout mice. HSCs transduced by the different viruses were transplanted into either lethally irradiated Rag-1-deficient animals or Artemis knockout mice treated with a nonmyeloablative dose of Busulfan. In both models, transplantation of HSCs transduced by a vector that used a murine phosphoglycerate kinase (PGK) promoter led to a complete functional correction of the immunodeficiency. Corrected animals displayed rescue of mature B cells with normal levels of serum immunoglobulins, together with complete rescue of the T cell compartment as evidenced by the presence of mature T lymphocytes in peripheral blood as well as normal values of thymocytes in thymus. Those B and T cells were capable of activation, as shown both by in vitro stimulation responses and in vivo after immune challenge. Overall, the results indicate that a gene therapy approach for RS-SCID involving the transplantation of genetically modified HSCs is indeed feasible. Furthermore, our studies suggest the possibility that nonmyeloablative conditioning regimens might be effectively used to promote engraftment of genetically modified cells in the case of diseases where standard irradiation-based myeloablative bone marrow transplantation protocols may prove problematic.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Lentiviral vector, transduction and expression of human Artemis. (A) Schematic representation of the pHAGE lentiviral construct used to express human Artemis cDNA from three different internal promoters, i.e., CMV, EF1α, and PGK. PBS-K, lysine tRNA-binding site; PSI, packaging signal; RRE, Rev responsive element; CPPT, central polypurine tract; WPRE, Woodchuck hepatitis posttranscriptional regulatory element; dU3, deleted U3. (B) Western blot analysis of lysates purified from 293 HEK cells transduced with lentiviral constructs expressing human Artemis. HEK 293, uninfected control. (C) Southern blot analysis of gDNA purified from bone marrow of transduced/transplanted mice. A representative sample from mice transplanted with cells transduced with lentiviruses expressing human Artemis (CMV, EF1α, and PGK) or GFP is shown. Increasing amounts of known plasmid vector DNA was used for copy number control. (D) RT-PCR of marrow samples as in C. Each group shows the undiluted and 1:3 diluted template reactions. GAPDH amplification serves as loading control.

Fig. 2.

Fig. 2.

B cell rescue in Rag-1 KO transplanted mice. (A) Rescue of B220+ IgM+ mature B cells in PB of transplanted mice over time. Recovery of mice receiving cells transduced with lenti-CMV (n = 4), lenti-EF1α (n = 6), lenti-PGK (n = 6), lenti-GFP control (n = 6), or cells from WT CD45.1 (n = 4) control is shown. (B) Representative FACS plots of individual mice transplanted with cells as in A. The presence of B220+ IgM+ mature B cells in PB and spleen (SP) is shown. (C) Representative pictures of spleens isolated from mice rescued by using cells transduced with lenti-PGK or -GFP or from mice transplanted with WT CD45.1 cells. (D) PCR analysis of genomic DNA purified from splenocytes was used to evidence the presence of V(D)J rearrangements. Each lane corresponds to an individual mouse. (E) IgM levels in serum of transplanted mice are shown. (F) IgG levels in serum of transplanted mice are shown. For details, see Materials and Methods.

Fig. 3.

Fig. 3.

T cell rescue in Rag-1 KO transplanted mice. (A) Rescue of CD4+ mature T cells (Left) and CD8+ mature T cells (Right) in PB of transplanted mice over time. Recovery of mice receiving cells transduced with lenti-CMV (n = 4), lenti-EF1α (n = 6), lenti-PGK (n = 6), lenti-GFP control (n = 6), or cells from WT CD45.1 control (n = 4) is shown. (B) Representative FACS plots of individual mice transplanted with cells as in A. PB cells and thymocytes were stained by using antibodies against CD4 and CD8.

Fig. 4.

Fig. 4.

Rescue of the immune system of Artemis-deficient mice. (A) Rescue of B220+ IgM+ mature B cells and CD4+/CD8+ mature T cells in PB of transplanted mice over time. Recovery of mice receiving cells transduced with lenti-PGK (n = 7), lenti-GFP (n = 3) or cells from WT CD45.1 (n = 3) control is shown. (B) Representative FACS plots of individual mice transplanted with cells as in A. The presence of B220+ IgM+ mature B cells and CD4+/CD8+ mature T cells in PB and spleen is shown. (C) Total number of lymphocytes in PB of transplanted mice 16 weeks after BMT compared with unmanipulated C57BL/6 mice (WT B6).

Fig. 5.

Fig. 5.

In vivo challenge and recovery of lymphocyte progenitors in Artemis KO rescued mice. (A) Relative levels of anti-keyhole limpets hemocyanin IgM (Left) or IgG (Right) antibodies in serum of mice receiving cells transduced with lenti-PGK or -GFP or cells from CD45.1 (n = 3 in all groups). Values represent levels of antibodies detected at 1:1,250 dilution of serum obtained either before (“pre”) or 28 days after challenge (“d28”). Data are expressed as mean values (±SEM) of triplicate samples. (B) Representative FACS plots of individual mice from the different groups. Bone marrow samples were analyzed for the presence of different B cell progenitors by using antibodies against IgM, B220, and CD43. Pregated IgM− cells were further defined as B220+ CD43+ (pro-B cells) or B220+ CD43− (pre-B cells). The red square denotes the percentage of pre-B cells in all of the different mice. (C) Representative FACS plots of individual mice transplanted as in B. Thymuses were stained by using antibodies against CD4 and CD8.

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