A small peptide derived from the HIV-1 gp120 glycoprotein forms positively charged fibrils that enhance transduction efficiencies of retro- and lentiviral vectors.
InstitutionsUKU. Institut für Medizinische Mikrobiologie und Hygiene
UKU. Institut für Molekulare Virologie
Low transduction efficiencies are a common problem in retro- and lentiviral gene transfer. Here, I have presented a new approach to boost retroviral gene transfer using self-assembled nanofibrils formed by the 12-amino acid peptide termed EF-C (Enhancement Fragment C). This peptide was derived from the HIV-1 external envelope glycoprotein gp120. The fibrils form instantaneously upon dilution of peptide in aqueous media and adopt a typical amyloid structure that was observed due to Thioflavin T and Congo Red staining and atomic force microscopy. Measurement of ζ-potential charge of aqueous solution of EF-C revealed that the nanofibrils have positively charged surface, supposed that their enhance infection by neutralizing the repulsion between the negatively charged viral and cellular membranes. My data obtained using confocal fluorescence microscopy shown that fibrils bind virions and increase their adsorption to cells and subsequent infection. Addition of EF-C to virus stocks or target cells boosted gene delivery into multiple cell types, including primary T cells, macrophages and CD34+ stem cells, and was substantially more effective than other reagents commonly used to increase transduction. Moreover, like RetroNection, the most commonly used reagent to increase retroviral transduction, EF-C potently boosts transduction also when immobilized on cell culture dishes. This effect was confirmed with retro- and lentiviral particles carrying various viral envelope proteins frequently used for gene transfer and therapy. The enhancing effect was independent of the viral glycoprotein but still required the respective cellular receptor(s). Furthermore, the fibrils allow a fast and convenient concentration of virions without the need of ultracentrifugation. The result presented in this thesis demonstrate that EF-C nanofibrils provide a convenient, flexible and effective means to increase retro- and lentiviral gene transfer in basic research and clinical applications.
Subject headings[GND]: HIV Envelope Protein gp120 | HIV-Infektion | Gentransfer | Enhancer
[LCSH]: Retroviruses | HIV (Viruses) | Gene transfer | Genetic transformation | Glycoproteins | HIV infections
[MeSH]: Glycoproteins | Retroviridae | HIV infections | Transformation, genetic
[Free subject headings]: Retro- and lentiviral gene transfer | Transduction of primary cells | Self-assembled nanofibrils | HIV-1 envelope glycoprotein gp120 | Enhancer of retroviral transduction
[DDC subject group]: DDC 610 / Medicine & health
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Please use this identifier to cite or link to this item: http://dx.doi.org/10.18725/OPARU-4106