Role of transgene regulation in ex vivo lentiviral correction of artemis deficiency

Abstract

Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID.

Figures

FIG. 1.

Lentiviral vector constructs and Artemis-associated cytotoxicity. (A) Lentiviral vectors were used for analysis and complementation of Artemis deficiency. Experimental lentiviral vectors were engineered to express the Artemis coding sequence under the transcriptional regulation of either the human elongation factor 1α promoter (EF1α), the human phosphoglycerate kinase promoter (PGK), or the 1-kilobase endogenous human Artemis promoter (APro). Lentiviral vectors serving as transduction controls expressed either green fluorescent protein (CSIIEG) or the puromycin resistance gene (CSII/E-Puro) regulated by EF1α. Abbreviations: CMV, cytomegalovirus early promoter/enhancer region; U3/U5/R, unique 3′/unique 5′/repeat regions of the HIV long terminal repeat; ψ, packaging signal; cPPT, central polypurine tract; WPRE, woodchuck posttranscriptional regulatory element. Arrows indicate sites and direction of transcript initiation. (B) Artemis regulation by APro avoids cytotoxicity. Murine NIH 3T3 cells were transduced at increasing MOIs using CSII/E-Puro, EF1α-Artemis, PGK-Artemis, or APro-Artemis lentiviral vectors as indicated. Cell survival was assessed 5 days posttransduction by MTT assay and plotted as the percentage of cells surviving in control, untreated populations. Each value represents the mean of three replicates+SD. (C) Low-level expression conferred by the Artemis promoter. Luciferase expression constructs were transfected into human 293T cells and then lysates were assayed for luciferase activity. The mean relative level of expression versus a cotransfected Renilla luciferase standard is shown±SD.

FIG. 2.

Donor engraftment in SCID-A mice: Time course. After infusion of transduced donor marrow, peripheral blood was collected over a period of 16 weeks to monitor donor lymphoid engraftment and repopulation, and plotted as the number of donor lymphocytes (×103) per microliter. Each symbol represents the results from a single animal for the groups indicated in the key. Bars represent mean values.

FIG. 3.

Repopulation of circulating lymphocyte subsets in transplanted SCID-A mice. Peripheral blood was collected over a period of 16 weeks posttransplantation for analysis of lymphocyte populations. (A) Circulating B lymphocytes (B220+NK1.1−), (B) helper T lymphocytes (CD3+CD4+), and (C) cytotoxic T lymphocytes (CD3+CD8+) are plotted over time as the number of lymphocytes×103 per microliter within the donor lymphocyte (CD45.1+) population. Percentages were obtained by flow cytometry and cell counts were obtained by Hemavet analysis of whole blood.

FIG. 4.

Functional in vivo immune response. (A and B) Artemis-treated animals as well as control C57BL/6 and SCID-A untreated animals were challenged with NP-KLH and boosted 5 weeks after the initial challenge. One week after the final boost, sera were collected and analyzed by ELISA for the presence of (A) IgM and (B) IgG elicited against NP-KLH as compared with naive sera collected prechallenge. Immunoglobulin levels are plotted as absorbance (450 nm) versus serum dilution. (C and D) Upon sacrifice of Artemis-treated, C57BL/6 control, and SCID-A control animals, splenocytes were prepared and then plated in the presence of increasing amounts of either (C) anti-CD3 or (D) concanavalin A. Proliferative indices were calculated by dividing the MTT absorbance acquired in the presence of mitogen by absorbance acquired from samples without mitogen stimulation.

FIG. 5.

Repopulation within lymphoid organs. Upon sacrifice, primary lymphoid organs were harvested and single-cell suspensions were analyzed by flow cytometry for the presence of B, helper T, and cytotoxic T donor lymphocyte populations of Artemis-treated animals, control C57BL/6 animals, and control SCID-A animals. (A) B lymphocyte development was characterized in the bone marrow, identifying both immature (B220+NK1.1−) and mature (B220+IgM+) B lymphocytes. (B) T lymphocyte development was monitored in the CD3+ donor lymphocyte compartment within the thymus, detecting the presence of helper T cells (CD4+), cytotoxic T cells (CD8+), double-positive T lymphocytes (CD4+CD8+), and double-negative T lymphocytes (CD4−CD8−). (C) Spleen and (D) lymph nodes were analyzed for the presence of B (B220+IgM+), helper T (CD3+CD4+), and cytotoxic T (CD3+CD8+) cells. Results are plotted as the percentage of total lymphocytes in control C57BL/6 animals and control untreated SCID-A animals, and as the percentage of total donor lymphocytes in animals receiving transduced bone marrow.

FIG. 6.

Lymphoid reconstitution after secondary transplantation of transduced marrow. Bone marrow was collected individually from primary recipients treated with (A) APro-Artemis, (B) PGK-Artemis, and (C) EF1α-Artemis (n=4) and infused into each of three secondary irradiated (800 rads, X-irradiation source) C57BL/6 recipient animals. Peripheral blood was collected at monthly time points posttransplantation. B lymphocyte (B220+NK1.1−) as well as helper T (CD3+CD4+) and cytotoxic T (CD3+CD8+) lymphocyte repopulations were found to persist in secondary transplant recipients, plotted as percentage of the donor lymphocyte compartment. Shaded circles represent lymphocyte percentages in secondary transplant recipients 16 weeks posttransplantation; open circles represent lymphocyte percentages in respective primary donors at week 16 after primary transplantation.