Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression

Abstract

MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp-targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence- and dose-dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF-α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus-mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis.

Adaptive Immune Responses Elicited by Baculovirus and Impacts on Subsequent Transgene Expression In Vivo

“…Bac-CE expressing enhanced green fluorescent protein (EGFP) as the reporter was constructed as described previously (30). To construct pBac-luc/w, the firefly luciferase gene was amplified from pGEM-luc (Promega) and cloned into pBac-CMV5 (whose polyhedrin promoter was replaced by the cytomegalovirus immediate-early [CMV-IE] promoter [31]). WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) was then cloned at the 3= end of the luciferase gene to yield pBac-luc/w.…”

“…pBac-T2Fluc/w was constructed by subcloning the CMV-IE-luciferase-WPRE cassette into pBac-T2 (17) between the inverted repeat/direct repeat (IR/DR) elements for Sleeping Beauty (SB) transposase recognition. pBac-SB100X encoding the SB100X transposase under the control of the CMV-IE promoter was constructed as described previously (31). pBac-luc/w, pBac-T2Fluc/w, and pBac-SB100X were used to generate the recombinant BV vectors Bac-luc/w, Bac-T2Fluc/w, and Bac-SB100X according to the instructions provided with the Bac-to-Bac system (Invitrogen).…”

Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study

“…The absence of fluorescent signals in the mock-transduced cells and the distinct signals associated with the chromosomes in H-d2E [a stable HEK293 clone harboring integrated d2egfp (18)] validated the accuracy of FISH. Consequently, the fluorescent signals outside the chromosomes in the BacCre + BacL-CdE/W-CEO group confirmed the episomal form of the recombined minicircles.…”

“…Conversely, targeted genome editing can be attained by using zinc finger nuclease (34) or transcription activator-like (TAL) effector nucleases (35), but these nucleases mediate integration at low efficiencies and require antibiotic selection of cells, which may preclude their uses in regenerative medicine because immediate stem cell implantation following genetic modification is desired. In the BV setting, attempts to incorporate adeno-associated virus (AAV) inverted terminal repeats (2,36) or Sleeping Beauty transposon (18,19) into BV vectors have been made. For instance, a hybrid BV exploiting the Sleeping Beauty transposon system was developed to extend the expression of anti-angiogenic factors for anti-cancer therapy (19).…”

“…However, one drawback of BV is that it typically enables transient expression for <7 days (14–16) unless antibiotic selection (17) or vector engineering for transgene integration is adopted (2,18,19), which may hinder its direct applications in some scenarios. For instance, ASCs have become a popular cell source for bone engineering, but ASCs are poorer than BMSCs in osteogenesis potential (10).…”