Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal...

Area of Science:

• * Molecular Biology
• * Virology
• * Gene Therapy Vector Development

Background:

• * Recombinant adenoviruses with deleted E1 genes exhibit transient transgene expression due to cellular or viral E1A-substituting activities. These activities lead to viral antigen synthesis and cytotoxic T-lymphocyte-mediated clearance of transduced cells.
• * The E4 region of adenoviruses significantly enhances viral gene expression at both transcriptional and posttranscriptional levels, contributing to the transient nature of transgene expression.

Purpose of the Study:

• * To develop a method for achieving stable transgene expression from recombinant adenoviruses by additionally deleting the E4 region.
• * To create a packaging cell line capable of complementing both E1 and E4 functions for the production of E1/E4 doubly defective adenoviruses.

Main Methods:

• * Cloning of the distal ORF6+ORF7 segment from the adenovirus type 5 E4 locus into a vector under the control of a dexamethasone-inducible mouse mammary tumor virus (MMTV) long terminal repeat (LTR).
• * Transfection of this construct into 293 cells to establish the IGRP2 packaging cell line.
• * Characterization of IGRP2 cells for their ability to support the growth of E1- and E4-defective adenoviruses, including plaque purification and propagation of recombinant viruses.

Main Results:

• * The IGRP2 cell line successfully rescues the growth defect of E1+ E4- adenoviral deletants, demonstrating functional complementation of both E1 and E4 regions.
• * DNA and RNA analysis confirmed that the MMTV promoter drives ORF6+ORF7 expression and allows alternative splicing, producing both ORF6/7 and ORF6 mRNAs.
• * IGRP2 cells exhibit extended cell confluence compared to parental 293 cells and facilitate the plaque purification of E1- or E4-defective viruses.
• * Helper-free propagation of recombinant lacZ-encoding doubly defective adenoviruses with various E4 deletions was achieved using IGRP2 cells. The integration of a limited E4 segment in IGRP2 cells is expected to impair the emergence of replication-competent particles.

Conclusions:

• * The novel IGRP2 packaging cell line enables the generation and propagation of E1 and E4 doubly defective adenoviruses.
• * This approach overcomes the transient transgene expression issue associated with conventional E1-deleted adenoviral vectors.
• * The developed system holds promise for improving the efficacy of gene therapy applications utilizing adenoviral vectors.