The Optimization of a Cost-Effective Lentiviral Vector Production Protocol for Animal Gene Therapy Trials
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Hetrick, Brian
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Abstract
This thesis describes a cost effective method for the mass production of lentiviral vector particles for the use in animal gene therapy trails. Viral gene therapy is the process by which genes can be introduced, integrated, or up regulated within the genetic messages of viral particles. Due to their relatively small genomes and there affinity for particular cell surface receptors, viral vectors can easily be manipulated to carry genes to a particular cell type. This potentially makes viral vectors a favorable new method to fight diseases. In order to produce replication deficient lentiviral particles safely in a laboratory, the genome, the envelope, and accessory proteins are split into multiple plasmids. Only those cells that have received all the necessary plasmids with all the required genes will form functioning viral particles. Transfections are a tedious and costly procedure that if not standardized will lead to varying results each time virus is produced. To find the most cost effective and efficient way to produce lentiviral particles, multiple transfections were preformed using three common chemical transfection reagents, LipofectamineTM 2000, calcium phosphate, and polyethylenime (PEI). The costs of the procedures and efficiency of the transfections were assed. It was found that the latter of the three methods was the most cost-efficient. In addition to reducing the cost of transfections, anion exchange and size exclusion will be used to amplify the titer and purity of the viral particles produced from these transfections will greatly reduce the need of multiple injections and larger volume of viral particles in animal trials.
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Viral gene therapy, Lentivirus, HIV, Transfection