Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted

Abstract

Adenovirus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems. Most Ad vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted E1 gene functions in trans. Unfortunately, the use of high titers of E1-deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors. We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions. The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA. We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome. The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.

FIG. 1

Diagrammatic representation of the steps used to isolate pBHG11Δpol (A), AdΔpol and AdΔpol/pBHG11 (B), and AdLacZΔpol (C). mu, map units.

FIG. 2

(A) DNA of each of the indicated viruses was digested with HindIII, electrophoresed, blotted, and probed with a ³²P-labeled dl7001 genomic probe. The recombinant viruses (AdΔpol/dl7001 and AdΔpol/pBHG11) both contained the Δpol alteration. This was verified by the demonstration that the 5.3-kb polymerase-encoding fragment normally present in dl7001 (location indicated by the solid arrowheads) migrated as a 4.7-kb DNA fragment in the Δpol-containing viruses (new location depicted by the open arrowhead). Note also that the pBHG11-derived virus contained a larger E3 deletion, (×), in contrast to the dl7001-derived E3 deletion (⋆). The indicated cell lines (2 × 10⁶ cells) were identically infected with AdΔpol/dl7001 at an MOI of ∼1.0 PFU/cell. Twenty-four hours after infection, total DNA was isolated, 4 μg of each was digested with HindIII; the fragments were electrophoretically separated, blotted onto a nylon membrane, and probed with ³²P-labeled dl7001 genomic DNA. Note the complete lack of replication of AdΔpol/dl7001 in 293 cells.

FIG. 3

B-6 cells were infected with AdLacZΔpol; total DNA was harvested 36 h later and digested with NotI and EcoRI, and the pattern of DNA fragments obtained was compared with that for NotI- and EcoRI-digested dl7001 DNA. The 6.5-kb left inverse terminal repeat (L-ITR)-E1-containing fragment of dl7001 (⋆) was replaced by the lacZ minigene cassette that generates three fragments of 3,879, 3,050 (), and 708 bp (not shown). The normal polymerase-coding region of dl7001 is contained within a 5,100-bp fragment (○) and is decreased in size to 4,492 bp (×) in Ad LacZΔpol. Std., size standards (positions are indicated in kilobases); CMV, cytomegalovirus.

FIG. 4

293 or B-6 cells (2 × 10⁶) were infected at an MOI of 0.01 BFU with Ad LacZΔpol, and the total BFU generated was assessed after limiting dilution infection and X-Gal staining of C-7 cells.

FIG. 5

(A) 293 or B-6 cells (2 × 10⁶) were infected at an MOI of 1.5 BFU with Ad LacZΔpol, and total DNA was harvested after the indicated incubation times. DNA was digested with HindIII, electrophoresed, blotted, and probed with ³²P-labeled dl7001 DNA. (B) HeLa cells (2 × 10⁶) were infected at the indicated MOIs with AdLacZΔpol or Adsub360LacZ and probed with ³²P-labeled dl7001 DNA as described for panel A. Densitometric analysis of the image was done with the NIH Image software package.

FIG. 6

(A) 293 or B-6 cells (2 × 10⁶) were infected at an MOI of 1.5 BFU with Ad LacZΔpol, and protein lysates isolated from the infected cells were electrophoresed, blotted, probed with a fiber polyclonal antibody, and visualized with the ECL system. Dilutions of the B-6 cell-derived lysates are indicated. (B) HeLa cells (2 × 10⁶) were infected at an MOI of 500 with AdLacZΔpol or Adsub360LacZ, and the fiber protein was visualized as described for panel A. Six micrograms of protein lysate from each infection was loaded per well. Each arrow indicates the location of the fiber-specific band, which correctly migrated as a 66-kDa protein based on comparison with the molecular weight standards included in the gel electrophoresis. To further verify that the bands indicated represent fiber protein monomer, the control lysate lane contained a portion of a protein lysate derived from the productive infection of B-6 cells with AdLacZΔpol. Note the lack of fiber expression in the lysate derived from AdLacZΔpol infection of HeLa cells.

FIG. 7

AdLacZΔpol (10⁹ BFU) was injected into the tail veins of BALB/c mice. Six days later, liver sections were harvested and stained for β-galactosidase activity. Note the stained nuclei present only in the infected cells (A) and the complete lack of β-galactosidase from a mock-infected age-matched control animal (B), confirming successful transduction and sustained transgene expression in vivo.