Integration of the hybrid adenoretroviral vector AdLTR-luc involves both MoMLV elements flanking the transgene

Abstract

Vector delivery is still a bottleneck for gene therapy. To overcome some disadvantages of adenoviral and retroviral vectors, we developed a hybrid vector. This hybrid vector, AdLTR-luc, was created by adding two elements from Moloney murine leukemia virus (MoMLV) flanking the luciferase cDNA into an E1/E3-deleted, replication deficient serotype 5 adenovirus vector (Zheng et al., Nature Biotechnol, 2000), and demonstrated that the MoMLV element upstream of the luciferase cDNA was broken during the integration event. The purpose of the current study was to determine if the MoMLV element downstream of the luciferase cDNA was also broken when integration occurred. We used the same A5 cell clones (#10 and 11) from the earlier the paper along with restriction endonuclease digestions, plus Southern hybridization, and PCR. Southern hybridization indicated that the luciferase cDNA was intact in the cloned cells. Results from Xho I and Sal I digestions showed that integration occurred in cloned cells. Southern hybridizations after Nco I digestion suggested that there was a break in both MoMLV elements, upstream and downstream of the luciferase cDNA. After DNA digestion with Not I, hybridization analyses indicated that the MoMLV upstream element was broken during integration. Digestion of genomic DNA with either Xba I/Kpn I, Bam HI/Sac I, or Bam HI/Nco I demonstrated that the MoMLV downstream element was also broken during integration. A PCR assay was unable to amplify the junctional region between the downstream MoMLV element and the adenoviral E2B gene, consistent with a break in that element. Although AdLTR-luc integration is atypical (Zheng et al., Nature Biotechnol, 2000), the present results suggest that both MoMLV elements have important roles in this event.

Keywords: Hybrid vector; LTR; adenoretrovirus; gene therapy; integration.

Figure 1

Structure of AdLTR-luc and Southern hybridization. A. Diagram of AdLTR-luc. AdLTR-luc contains the MoMLV elements described in the text: 2.7 kb upstream of the luciferase cDNA and 1 kb downstream. Luciferase served as a reporter gene, and the SV40 polyadenylation sequence was downstream of the luciferase cDNA. See text for details on construction. B. Luciferase activity from clones #10 and 11. C. Partial diagram of Bam HI and Not I target sites in AdLTR-luc. D. Southern hybridization to assess the integrity of the luciferase cDNA. DNA samples were obtained from cloned A5 cells (#s 10, 11) transduced with AdLTR-luc, or from non-transduced cells (N). Positive control (P) DNA was from uncloned cells transduced with AdLTR-luc 2 days before harvesting DNA. Each lane represents 15 µg of DNA applied and hybridized with the luciferase probe as described in the text. Note that cloned cell samples showed the same band (2.7 kb) as the positive control sample. At the left side of panel D, the migration positions of marker DNA (M; 1 Kb DNA ladder, GIBCO BRL, Rockville, MD) are shown in base pairs (bp).

Figure 2

Southern hybridization to assess the presence of break points in either the 2.7 kb or 1 kb of MoMLV elements. All DNA samples were as in Fig. 1D. Samples were digested with Xho I, Sal I and and Nco I and hybridized with the luciferase probe. Panel A is a partial diagram of enzyme target sites in AdLTR-luc. Panel B shows Southern hybridization results. The migration positions of marker DNA (M) in base pairs (bp) are shown at the left.

Figure 3

Southern hybridization to assess the presence break points in the 2.7 kb MoMLV element. All samples were as in Fig. 2B. Samples were digested with Not I and hybridized with the luciferase probe. Panel A is a partial diagram of enzyme target sites in AdLTR-luc. Panel B shows Southern hybridization results. The migration positions of marker DNA (M) in base pairs (bp) are shown.

Figure 4

Southern hybridization to determine the presence of break points in the 1 kb MoMLV element. All samples were as in Fig. 2B. Samples were digested with Xba I/Kpn I, Bam HI/Sac I or Bam HI/Nco I and hybridized with the luciferase probe. Panel A is a partial diagram of enzyme target sites in AdLTR-luc. Panel B shows Southern hybridization results. The migration positions of marker DNA (M) in base pairs (bp) are shown.

Figure 5

PCR assay to assess break points in the 1 kb MoMLV element. Panel A shows the design of PCR primers used to amplify the junction between the 3´LTR and the E2B region (PCR 1, 2 and 3) in AdLTR-luc. Panel B shows results of a PCR sensitivity assay. Panel C shows the two negative control assays used. For the non-transduced A5 cell genomic DNA samples, 0.5 µg DNA was used. Panel D shows results from cloned A5 cell samples (#10 and 11; 10 µg template DNA was used). Positive controls (P; 500 ng template AdLTR-luc DNA was used). The migration positions of marker DNA (M) in kilobase pairs (kb) are shown. The PCR assays used to detect amplicons 1, 2 and 3 are indicated by numbers 1, 2 and 3 in each corresponding lane of panels B - D.