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Recombinant LuIII autonomous parvovirus as a transient transducing vector for human cells
I H Maxwell1, F Maxwell, S L Rhode
1University of Colorado Cancer Center, Denver 80262.
Human Gene Therapy
|August 1, 1993
Summary
Autonomous parvoviruses like LuIII can be engineered for gene transfer. Recombinant LuIII viruses carrying reporter genes successfully transduced cells, showing potential for gene therapy applications requiring transient expression.
Area of Science:
- Molecular Virology
- Gene Therapy Vectors
- Recombinant DNA Technology
Background:
- Autonomous parvoviruses, exemplified by LuIII, possess a unique replication strategy.
- Engineering parvovirus genomes offers potential for novel gene delivery systems.
- Reporter gene expression is a standard method for assessing viral vector efficacy.
Purpose of the Study:
- To construct and characterize recombinant autonomous parvoviruses based on the LuIII genome.
- To evaluate the gene transfer capabilities of these engineered parvoviruses using reporter genes.
- To assess the potential of these vectors for applications such as gene therapy.
Main Methods:
- Construction of infectious clones of LuIII (pGLu883) with reporter genes (luciferase, beta-galactosidase) replacing viral coding sequences.
- Co-transfection of cells with recombinant constructs and helper plasmids providing viral proteins.
- Assay of transducing virions in culture medium and reporter gene expression in recipient cells.
- Neutralization assays using antiserum to LuIII.
- Optimization of vector production by minimizing homology to prevent replication-competent virus generation.
Main Results:
- Recombinant LuIII genomes underwent excision and replication upon co-transfection.
- Transducing virions carrying reporter genes were produced and detected in culture media.
- Transducing activity was neutralized by anti-LuIII antiserum, confirming viral origin.
- Efficient production of replicative form DNA and transducing virions was observed in HeLa, 293, and NB324K cells.
- Minimizing homology in helper constructs reduced replication-competent virus production, yielding pure reporter virus preparations.
- Luciferase expression was observed in recipient cells for up to 7 days.
- Both viral DNA strands were packaged, with a predominant (-) strand.
Conclusions:
- Engineered autonomous parvoviruses, such as LuIII, can serve as effective gene transfer vectors.
- These vectors are capable of delivering reporter genes and achieving transient expression in recipient cells.
- Autonomous parvoviruses hold promise for gene therapy applications, particularly when transient gene expression is desired.