ARTIFICIAL VIRUSES · Design of artificial viruses by combinatorial protein engineering
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2004-06-01 → 2005-05-31
- EU contribution
- €40,000
- Participants
- 1
- Scheme
- ERG
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Results in brief
Final Activity Report Summary - ARTIFICIAL VIRUSES (Design of artificial viruses by combinatorial protein engineering)
With this research project we aimed at developing a high-throughput screening method for the evolutionary selection of protein-based non-viral gene delivery systems that make use of the cell-entry mechanisms of bacterial protein toxins. The entire project, which was scheduled to last two years, involved generation of gene libraries encoding for chimeric DNA-carrier proteins, expression of these gene libraries inside the aqueous compartments of a water-in-oil emulsion and subsequent selection of protein-DNA complexes for their capacity to transfect mammalian cells in culture. One year after the start of the project, we have managed to design, clone and express the chimeric proteins mentioned in the initial project description. Unfortunately, expression levels of the chimeric proteins inside the w/o emulsions was rather low and needed optimisation. We have spent a great deal of our time optimising the S30 extracts and reaction mixtures used for transcribing and translating the gene constructs inside emulsions, which turned out to be worthwhile. The expression levels we have now reached are high enough to continue the work on selecting chimeric proteins for their capacity to facilitate the cell entry of associated pDNA. The coming year we hope to finalise the selection experiments and demonstrate the in vitro compartmentalisation can be used for the screening and selection of artificial viruses.
Data: CORDIS, © European Union
Project objective
The specific delivery of therapeutic genes to defined target cell populations is a major challenge in gene therapy. This requires a sophisticated vector that is able to protect the DNA from degradation, interact specifically with cell surface receptors on target cells, cross the cell membrane and allow nuclear import of the exogenous DNA. Viral vectors are very efficient in depositing exogenous DNA into cells, but safety issues, lack of cell specificity, limited DNA packaging capacity and costs of large-sca le production hamper the general use of viral vectors for gene therapeutic application. Here, we propose to design artificial viruses" that are as efficient as viral vectors in gene delivery but are safe to use, cell-type specific and can be cost-effectiv ely produced at large scale. These artificial viruses will be designed to utilize the cellular entry mechanism of bacterial protein toxins to deliver DNA into target cells. A "survival of the fittest" approach will be used to select for artificial viruses most capable of transfecting plasmid DNA into mammalian cells from a large DNA library encoding for many different artificial viruses. This will be achieved by generating libraries of hybrid genes that combine the cell surface receptor-binding domain and t ranslocation domain of several bacterial toxins with the DNA-binding domain and nuclear localization signal of several transcription factors. Single members of the hybrid gene libraries will be transcribed and translated inside micron-sized aqueous droplet s of a water-in-oil emulsion (the artificial cells). Multiple copies of identical chimeric proteins that are properly folded will self-assemble into an artificial virus inside the artificial cell by interaction of the DNA-binding domain of the chimeric pro teins with the plasmid DNA. This creates a physical linkage between genotype (plasmid DNA) and phenotype (chimeric protein). After isolation of the artificial viruses #"
Original text from CORDIS.
Participants
- DEPARTMENT OF PHARMACEUTICS · UTRECHTCoordinatorCity levelNetherlands
Links
Data: CORDIS, © European Union