Date of Degree
MS (Master of Science)
Wendy J. Maury
Gene delivery via lentiviruses can yield long term expression of transgenes. Specificity of host cell targeting by viral vectors occurs primarily through viral glycoprotein (GP)/cellular receptor interactions. Ebola virus (EBOV) GP has broad tropism for a variety of cell types making this viral GP a potentially useful reagent for delivery of gene therapy. However, titers of EBOV GP pseudotyped lentiviruses are insufficient for practical use in clinical applications. Enhancement of EBOV-GP pseudotyped titers by as little as half a log might yield clinically applicable titers.
In an alanine scanning study, we identified 19 residues in EBOV-GP1 that increased transduction efficiency two to three fold. When mapped onto the crystal structure of EBOV GP, these residues were primarily located at the interface of GP1/GP2 suggesting these residue substitutions may confer conformational changes in the protein structure thereby enhancing transduction efficiency. To determine if combinations of these alanine substitutions might further enhance transduction, we have introduced the changes into EBOV GP in a stepwise manner. To date, introduction of some combinations of alanine substitutions resulted in as much as an eight-fold increase in transduction over WT GP, this being our super mutant combination, whereas other combinations eliminated transduction.
Identification of 5 additional mutations via 3D modeling of the glycoprotein uncovered an additional mutation in GP2, located at the GP1/GP2 interface, which also enhances EBOV GP transduction. Transduction of cell lines important for gene therapy including hepatocytes and porcine airway cells confirmed an enhancement in transduction as well. Other cell populations, specifically fibroblasts and renal cells, were also transduced but enhanced transduction was not observed indicating this phenomenon may be cell type specific. The in vivo studies were inconclusive because no expression was detected from any of the EBOV GP pseudovirions. Even expression of the positive control, GP64 particles, waned after 3 weeks post inoculation indicating insufficient quantities or poor quality pseudovirions were used. These EBOV GPs should prove useful for future gene therapy studies by providing an alternate glycoprotein that is as effective as GP64 at producing high titer lentiviral vectors.
Copyright 2009 Lindsay Marie Sandersfeld