Simple viral/minimal piggyBac hybrid vectors for stable production of self-inactivating gamma-retroviruses

Abstract

Background Transient production of gamma-retroviruses, including self-inactivating (SIN) retroviruses, is a common method for rapidly generating virus capable of gene delivery. Stable (continuous) production of virus is preferable to transient production for clinical and biotechnology purposes, however, because it allows for significant quantities of a uniform virus to be generated over a prolonged period of time, thus allowing for longitudinal functional studies and quality analysis. Unfortunately, stable production of SIN retroviruses is difficult to achieve. ResultsWe describe a novel method to rapidly and cost-effectively create packaging cells capable of continuously producing self-inactivating gamma-retroviruses. We imbedded the SIN proviral construct into a minimal piggyBac transposon vector and then integrated the hybrid vector into packaging cells that already stably expressed the viral gag-pro-pol and envelope genes. Cells that stably produced self-inactivating gamma-retroviruses could be identified (and purified) as early as 3 weeks after initial transfection; these cells produced virus for at least 9 weeks without a decline in titer.ConclusionsThis viral-minimal piggyBac hybrid vector allowed for the rapid generation and purification of packaging cells capable of stably producing self-inactivated gamma-retroviruses. This method can be applied to the large-scale production of viruses for use in research, biotechnology, and potentially, clinical trials.

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

“…In previous studies, we detailed its construction and demonstrated its ability to transpose cells as efficiently as full-length piggyBac transposons 11 ; we also demonstrated that this vector can be used for the rapid generation and purification of packaging cells capable of stably producing self-inactivated gamma-retroviruses. 26 Here, we show that this vector can be used to successfully integrate a 15 kb transgenic sequence into target cells, twice the capacity of retroviral vectors which are currently the most popular method for stable gene delivery. 27 While the integration efficiency of the minimal piggyBac system decreased as the size of the delivered insert increased, the largest transposon was still successfully integrated in 5% of transfected HEK293 cells making these vectors an attractive option for gene delivery.…”

Advances and challenges in gene therapy strategies for pediatric cancer: a comprehensive update

“…An example is the advent of self-inactivating vectors that reduce the oncogenic risks associated with retroviruses and lentiviruses. Similarly, third-generation adenoviral vectors are designed to minimize unwanted immune activation ( Troyanovsky et al, 2015 ; Shaw and Suzuki, 2019 ).…”