Engineering Enhanced Retroviral Vectors Through The Insertion Of A His6 Tag Into Vsv-g

Engineering of retroviral vectors by inserting novel protein or peptide sequences into viral proteins could readily improve their properties as gene therapy vehicles. Enhancements to VSV-G, an envelope protein commonly used to pseudotype retroviral and lentiviral vectors, could aid in vector purification and cell-specific targeting. Because the structure of VSV-G is unknown, it is difficult to rationally determine where to insert a new sequence without disrupting the virus? ability to transduce cells. We have developed a system to modify viral genomes through the random insertion of small peptides that may confer novel function to the viral vector. This high throughput method was used to generate a library of VSV-G mutants that have a His6 tag randomly incorporated at as many or all possible points in the original protein sequence. We have screened these libraries to isolate variants that can be purified by immobilized metal affinity chromatography while still retaining their ability to transduce cells. Purification by this method may provide advantages over current techniques in purity and scalability. In addition, these mutants will assist in identifying possible sites for the insertion of targeting ligands. The detailed genetic footprint provided by the comprehensive coverage of insertion sites will also provide further knowledge of the structure and function of VSV-G. Finally, we are utilizing this novel system to explore other desirable improvements to gene therapy vectors such as enhanced vector trafficking and antibody evasion. Craig Kennedy

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