Biosafety Assessment of Human Mesenchymal Stem Cells Engineered by Hybrid Baculovirus Vectors

Abstract

Mesenchymal stem cells (MSCs) hold promise for cell therapy, and implantation of MSCs engineered with a baculovirus transiently expressing the growth factor can augment the bone repair. To prolong the baculovirus-mediated transgene expression, we developed hybrid baculovirus vectors exploiting the FLP/Frt-mediated recombination for circular episome formation. Transduction of human MSCs with the hybrid baculovirus vectors harboring the osteoinductive bmp2 gene substantially extended the BMP2 expression and improved the cellular osteogenic differentiation. To confirm the potential in the clinical setting, the present study evaluated the biosafety profile of human MSCs engineered by the hybrid vectors. We unraveled that transduction of MSCs with the hybrid baculovirus vectors slightly impeded the cell proliferation after transduction, probably due to the perturbation of cellular gene expression and induction of innate responses. Nonetheless, the hybrid baculovirus vectors did not compromise the cell viability and cellular differentiation. No transgene integration into the host chromosome and disruption of the karyotype of the MSCs were observed. Additionally, no upregulation of proto-oncogenes or downregulation of tumor suppressor genes occurred in the MSCs transduced with the hybrid baculovirus vectors. Neither did the transduced MSCs induce tumor formation in nude mice. This study not only supported the safety of MSCs for cell therapy but also implicated the potential of the human MSCs engineered by the hybrid baculovirus vectors for their applications in clinical scenarios necessitating sustained transgene expression.

Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy

“…In contrast, no fluorescent spots were associated with the chromosome in the cells transduced with an egfp-bearing baculovirus deficient in the IR/DR elements and CMV-SB cassette. 20 Figure 2 thus collectively indicated that the transgene persistence stemmed from gene integration into the host chromosome, thanks to the SB-mediated transposition.…”

“…28 The CMV-EGFP cassette was also PCR amplified from pEGFP-N1 (Clontech, Mountain View, CA, USA) and subcloned into the oriP/EBNA1-containing pRep4 (Invitrogen, Carlsbad, CA, USA). The cassette comprising CMV-EGFP and oriP/EBNA1 was then subcloned into pFastBacDpolhDp10, whose polyhedrin and p10 promoters were removed from pFastBac DUAL 20 to yield pBac-COE-CE. To generate pBac-ITR-CE, the egfp gene from pEGFP-N1 was PCR amplified and subcloned into pAAV-MCS (Stratagene, Santa Clara, CA, USA) in between the CMV-IE promoter and the polyadenylation signal (pA).…”

“…48 The FISH experiments were performed as described with minor modifications. 20 The mitotic cells were prepared at 24 d.p.t. by colcemid (10 mg ml À1 , Sigma) treatment for 2 h, transferred to microfuge tubes, incubated in the hypotonic KCl solution (0.075 M) at 37 1C for 20 min and fixed in methanol/acetic acid (3:1).…”

“…19,20 Currently, a number of systems, including the oriP/Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1), AAV inverted terminal repeats (ITRs) and Sleeping Beauty (SB) transposon, are available to maintain the transgene in an episomal or integrated form. oriP is an origin of replication derived from Epstein-Barr virus, which can be recognized by EBNA1, leading to the replication and subsequent segregation of episomal Epstein-Barr virus DNA; 21 hence, constructs containing oriP/EBNA1 have been exploited for persistent expression.…”

Defective Antiviral Responses of Induced Pluripotent Stem Cells to Baculoviral Vector Transduction

“…For proliferation assays, iPSCs cultured in 96-well plates were transduced and analyzed at 3 days posttransduction (dpt) using a bromodeoxyuridine (BrdU) cell proliferation assay kit (Millipore) as described previously (8). The apoptosis of iPSCs was quantified by two-dimensional flow cytometry, using an Annexin V-PE (phycoerythrin) Apoptosis Detection Kit I (BD Biosciences).…”