Characterization of the human artemis promoter by heterologous gene expression in vitro and in vivo

Abstract

Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B- T- radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5' rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells.

Figures

FIG. 1.

5′ upstream sequence of the human Artemis gene. The 1 kb genomic region located directly upstream of the human Artemis translational start site (TSS) was isolated by polymerase chain reaction amplification (nucleotide sequence shown). The region was analyzed using the online transcription factor binding site search engine TFSEARCH. Several potential transcriptional regulation sites were identified and are labeled according to their position relative to the human Artemis TSS. Transcription factor binding sites are in bold and are underlined. Canonical sequences are displayed directly above the APro sequence. The 5′ termini of APro deletion constructs relative to the TSS are shown boxed in black. Transcriptional start sites within the APro region were identified by 5′ rapid amplification of cDNA ends (RACE), clustered into four groups (see Figure 3), and the average start site location of each group is labeled and highlighted in light gray. A potential 5′ splice site located at −501 is italicized.

FIG. 2.

In vitro deletion analysis of the human APro region. (A) HEK 293T cells and (B) BJAB lymphoid cells were transfected with reporter constructs consisting of firefly luciferase cDNA regulated by APro, an APro deletion construct, the CAGS promoter (pKT2/Cal), or a promoterless control. Each cell population was cotransfected with a CMV regulated Renilla luciferase encoding plasmid to control for transfection efficiency. Luciferase expression was assayed and plotted as the ratio of firefly RLU/Renilla RLU as described in Materials and Methods.

FIG. 3.

Transcriptional start sites in the APro region mapped by 5′ RACE. 5′ RACE was performed on RNA isolated from (A) BLIN K− cells, (B) BLIN K+ cells, (C) BJAB cells, and (D) human liver. Each 5′ RACE product is displayed as a hatched bar positioned along the axis labeled with numbers representing nucleotide position upstream of the human Artemis TSS. The number of stacked hatched bars represents the number of 5′ RACE products identified at that particular nucleotide position: 3, 2, and 1 stacked bars represent 3, 2, and 1 5′ RACE products, respectively. 5′ RACE products are grouped into four regions and are boxed in light gray.

FIG. 4.

APro mediates low levels of green fluorescent protein (GFP) expression after lentiviral transduction in vitro. Mouse 3T3 cells were transduced with increasing amounts (0.3, 1, or 3 μL) of either (A) an EF1α regulated GFP lentiviral vector (CSIIEG) or (B) an APro regulated lentiviral vector (APro-GFP) and analyzed by flow cytometry for GFP expression. Dot plot analysis reveals a similar transduction frequency for each vector; however, CSIIEG transduced cells expressed GFP at a mean fluorescence intensity (MFI) significantly higher than APro-GFP transduced cell populations. (C) Histograms displaying the MFI exhibited by 3T3 cells transduced with 1000 μL of either CSIIEG or APro-GFP lentiviral supernatant reveal that cells transduced with CSIIEG exhibited an MFI of 216, whereas cells transduced with APro-GFP exhibited a much lower MFI of 21.5. CMV, cytomegalovirus early promoter/enhancer region; U3/U5/R, unique 3′/unique 5′/repeat regions of the HIV long terminal repeat; WPRE, woodchuck post-transcriptional regulatory element.

FIG. 5.

APro mediated GFP expression in vivo. Donor CD45.1 C57BL/6 bone marrow was transduced with either (A) an EF1α regulated GFP lentiviral vector (CSIIEG) or (B) an APro regulated lentiviral vector (APro-GFP) and transplanted into recipient CD45.2 C57BL/6 animals preconditioned with 800 rads. GFP expression was monitored by flow cytometry over a 16-week time period and is indicated for individual animals, represented by open circles, with mean values represented as solid black bars, both in units of percentage GFP positive cells in peripheral blood donor lymphocytes. (C) Four months post-primary transplant, animals were sacrificed and total bone marrow was infused into secondary C57BL/6 recipients. Four months post-secondary transplant, GFP expression mediated by the APro-GFP vector was observed to persist in secondary transplant recipients. (D) GFP expression mediated by the CSIIEG vector and by the APro-GFP vector was observed in both myeloid and lymphoid lineages. Subpopulations are graphed as percentages of the total GFP+ transduced donor (CD45.1) population.