Viral vector construction, production and vector-mediated gene transduction.

摘要

Until now, viral vectors are considered necessary for gene therapy, and current approaches are prohibited from wide applications mainly due to low efficiency and genotoxicity. The use of optimized vector production systems, the right choice of target cells, and improved transduction protocols may overcome these obstacles. To improve viral vector production, I initially optimized a calcium phosphate-mediated transfection method through inclusion of dextran and combined use of polybrene, and significantly improved the quality and quantity of the produce. Following that, multiple strategies, including a novel E. coli-based recombination system, Taq DNA polymerase treatment, and introduction of a bacteria toxic gene, were established and significantly improved the efficiency of generation of recombinant adenovirus vector. Moreover, multiple molecular manipulative strategies tested to a prototype retroviral vector system improved vector titers by 2-3 logs and led to enhanced transduction of a broad variety of cell types, especially cells of human and mouse haematopoietic and lymphocytic lineages that hold potential for gene therapy against a wide range of inherited and acquired diseases. Furthermore, a series of mutant tRNALys3 genes were constructed and expressed using the optimized viral vector production systems, and showed potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication through improved priming of HIV-1 reverse transcription from their targeting sites. Transduction of multiple copies of mutant tRNA Lys3 further enhanced the anti-HIV-1 potency. Lastly, a soluble tumor necrosis factor-α receptor (sTNFR)-Fc fusion protein was designed and expressed to meliorate neurons through neutralizing TNF-α. TNF-α-binding activity of secreted sTNFR-Fc from transduced cells was demonstrated and conditioned medium containing sTNFR-Fc was shown to be protective to neuronal cells from TNF-α-, HIV-1 Tat-, and gp120-mediated neurotoxicity. Overall, this study established multiple strategies and methods for improved viral vector production to facilitate gene therapy tests against HIV/AIDS and other diseases. The mutant tRNALys3- and sTNFR-Fc-based anti-HIV/NeuroAIDS strategies laid the groundwork for development of novel therapeutics against HIV and NeuroAIDS. Particularly, high efficiency transduction of cells of haematopoietic and lymphocytic lineages hold potential of using the genetically modified cells as non-invasive vehicles to deliver therapeutic substances across the blood-brain barrier into the central nervous system.