We recently developed hybrid baculovirus (BV) vectors that exploited FLPo/Frt-mediated DNA minicircle formation. Engineering of adipose-derived stem cells (ASCs) with the FLPo/Frt-based BV vectors enabled prolonged transgene expression and, after cell implantation into rabbits, ameliorated cartilage regeneration and bone repair. To translate the hybrid BV one step further toward clinical applications, here we assessed the biosafety profiles of the hybrid BV-engineered human ASCs (hASCs) in vitro and evaluated the immune responses elicited by the engineered porcine ASCs (pASCs) in large animals. We confirmed that the hybrid BV did not compromise the hASCs viability, immunosuppressive capacity, and surface characteristics. Neither did the hybrid BV cause chromosomal abnormality/transgene integration in vitro nor did it induce tumorigenicity in vivo. In the large animal study, pASCs were engineered with the hybrid BV expressing bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) and implanted into femoral bone defects in mini pigs. The hybrid BV-engineered pASCs enabled prolonged BMP2/VEGF expression and triggered the healing of massive segmental bone defects, while only eliciting transient antibody, cytokine, and local cellular immune responses stemming from the implantation procedure itself. These data altogether demonstrated the safety of the hybrid BV vectors for ASCs engineering and bone healing in large animals, hence implicating the potential in clinical applications.
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References
1.
RadaT., ReisR.L., and GomesM.E.Adipose tissue-derived stem cells and their application in bone and cartilage tissue engineering. Tissue Eng Part B Rev, 15, 113, 2009.
2.
VirkM.S., ConduahA., ParkS.H., LiuN., SugiyamaO., CuomoA., KangC., and LiebermanJ.R.Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone, 42, 921, 2008.
3.
KumarS., WanC., RamaswamyG., ClemensT.L., and PonnazhaganS.Mesenchymal stem cells expressing osteogenic and angiogenic factors synergistically enhance bone formation in a mouse model of segmental bone defect. Mol Ther, 18, 1026, 2010.
4.
ZouD., ZhangZ., HeJ., ZhangK., YeD., HanW., ZhouJ., WangY., LiQ., LiuX., ZhangX., WangS., HuJ., ZhuC., ZhangW., zhouY., FuH., HuangY., and JiangX.Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs. Biomaterials, 33, 2097, 2012.
5.
KostT.A., CondreayJ.P., and JarvisD.L.Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol, 23, 567, 2005.
6.
WangK.-C., WuJ.-C., ChungY.-C., HoY.-C., ChangM.D., and HuY.-C.Baculovirus as a highly efficient gene delivery vector for the expression of hepatitis delta virus antigens in mammalian cells. Biotechnol Bioeng, 89, 464, 2005.
7.
MerrihewR.V., ClayW.C., CondreayJ.P., WitherspoonS.M., DallasW.S., and KostT.A.Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells. J Virol, 75, 903, 2001.
8.
CondreayJ.P., WitherspoonS.M., ClayW.C., and KostT.A.Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proc Natl Acad Sci USA, 96, 127, 1999.
9.
HuY.-C., TsaiC.-T., ChangY.-J., and HuangJ.-H.Enhancement and prolongation of baculovirus-mediated expression in mammalian cells: focuses on strategic infection and feeding. Biotechnol Prog, 19, 373, 2003.
10.
AirenneK.J., HuY.-C., KostT.A., SmithR.H., KotinR.M., OnoC., MatsuuraY., WangS., and Yla-HerttualaS.Baculovirus: an insect-derived vector for diverse gene transfer applications. Mol Ther, 21, 739, 2013.
11.
ChenC.-Y., LinC.-Y., ChenG.-Y., and HuY.-C.Baculovirus as a gene delivery vector: Recent understandings of molecular alterations in transduced cells and latest applications. Biotechnol Adv, 29, 618, 2011.
12.
LinC.-Y., LuC.-H., LuoW.-Y., ChangY.-H., SungL.-Y., ChiuH.-Y., and HuY.-C.Baculovirus as a gene delivery vector for cartilage and bone tissue engineering. Curr Gene Ther, 10, 242, 2010.
13.
LuC.-H., ChangY.-H., LinS.-Y., LiK.-C., and HuY.-C.Recent progresses in gene delivery-based bone tissue engineering. Biotechnol Adv, 31, 1695, 2013.
14.
LinC.-Y., ChangY.-H., LinK.-J., YenT.-Z., TaiC.-L., ChenC.-Y., LoW.-H., HsiaoI.-T., and HuY.-C.The healing of critical-sized femoral segmental bone defects in rabbits using baculovirus-engineered mesenchymal stem cells. Biomaterials, 31, 3222, 2010.
15.
GodinoC., BriguoriC., AiroldiF., ToiaP., SaoliniM., FerrariA., CeraM., FragassoG., ImrosM.A., SalomoniM., TodeschiniP., GajateA.M., GianolliL., OppizziM., CapogrossiM.C., CondorelliG., and ColomboA.[Cardiac stem cell therapy for the treatment of chronic stable angina refractory to conventional therapy. State of the art and current clinical experience of the San Raffaele Hospital of Milan, Italy]. G Ital Cardiol (Rome), 12, 198, 2011.
16.
GlassG.E., ChanJ.K., FreidinA., FeldmannM., HorwoodN.J., and NanchahalJ.TNF-α promotes fracture repair by augmenting the recruitment and differentiation of muscle-derived stromal cells. Proc Natl Acad Sci USA, 108, 1585, 2011.
17.
SungL.Y., ChenC.L., LinS.Y., HwangS.M., LuC.H., LiK.C., LanA.S., and HuY.C.Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study. Nucleic Acids Res, 41, e139, 2013.
18.
LuC.-H., YehT.-S., YehC.-L., FangY.-H.D., SungL.-Y., LinS.-Y., YenT.-C., ChangY.-H., and HuY.-C.Regenerating cartilages by engineered ASCs: Prolonged TGF-β3/BMP-6 expression improved articular cartilage formation and restored zonal structure. Mol Ther, 22, 186, 2014.
19.
LiaoY.-H., ChangY.-H., SungL.-Y., LiK.-C., YehC.-L., YenT.-C., HwangS.-M., LinK.-J., and HuY.-C.Osteogenic differentiation of adipose-derived stem cells and calvarial defect repair using baculovirus-mediated co-expression of BMP-2 and miR-148b. Biomaterials, 35, 4901, 2014.
20.
QiuM.-T., HuJ.-W., YinR., and XuL.Long noncoding RNA: an emerging paradigm of cancer research. Tumor Biol, 34, 613, 2013.
21.
YehT.-S., Dean FangY.-H., LuC.-H., ChiuS.-C., YehC.-L., YenT.-C., ParfyonovaY., and HuY.-C.Baculovirus-transduced, VEGF-expressing adipose-derived stem cell sheet for the treatment of myocardium infarction. Biomaterials, 35, 174, 2014.
22.
SungL.-Y., ChenC.-L., LinS.-Y., LiK.-C., YehC.-L., ChenG.-Y., LinC.-Y., and HuY.-C.Efficient gene delivery into cell lines and stem cells using baculovirus. Nat Protocols, 9, 1882, 2014.
23.
GhildiyalM., SeitzH., HorwichM.D., LiC., DuT., LeeS., XuJ., KittlerE.L.W., ZappM.L., WengZ., and ZamoreP.D.Endogenous siRNAs derived from transposons and mRNAs in drosophila somatic cells. Science, 320, 1077, 2008.
24.
LinC.-Y., LinK.-J., LiK.-C., SungL.-Y., HsuehS., LuC.-H., ChenG.-Y., ChenC.-L., HuangS.-F., YenT.-C., ChangY.-H., and HuY.-C.Immune responses during healing of massive segmental femoral bone defects mediated by hybrid baculovirus-engineered ASCs. Biomaterials, 33, 7422, 2012.
25.
WolbankS., PeterbauerA., FahrnerM., HennerbichlerS., van GriensvenM., StadlerG., RedlH., and GabrielC.Dose-dependent immunomodulatory effect of human stem cells from amniotic membrane: a comparison with human mesenchymal stem cells from adipose tissue. Tissue Eng, 13, 1173, 2007.
26.
OgawaK., HiraiM., KatsubeT., MurayamaM., HamaguchiK., ShimakawaT., NaritakeY., HosokawaT., and KajiwaraT.Suppression of cellular immunity by surgical stress. Surgery, 127, 329, 2000.
27.
PatschC., PeitzM., OtteD.M., KesselerD., JungverdorbenJ., WunderlichF.T., BrustleO., ZimmerA., and EdenhoferF.Engineering cell-permeant FLP recombinase for tightly controlled inducible and reversible overexpression in embryonic stem cells. Stem Cells, 28, 894, 2010.
28.
ChenG.-Y., ShiahH.-C., SuH.-J., ChenC.-Y., ChuangY.-J., LoW.-H., HuangJ.-L., ChuangC.-K., HwangS.-M., and HuY.-C.Baculovirus transduction of mesenchymal stem cells triggers the toll-like receptor 3 (TLR3) pathway. J Virol, 83, 10548, 2009.
29.
ChuangC.K., WongT.H., HwangS.M., ChangY.H., ChenG.Y., ChiuY.C., HuangS.F., and HuY.C.Baculovirus transduction of mesenchymal stem cells: in vitro responses and in vivo immune responses after cell transplantation. Mol Ther, 17, 889, 2009.
30.
NovitskiyS.V., RyzhovS., ZaynagetdinovR., GoldsteinA.E., HuangY., TikhomirovO.Y., BlackburnM.R., BiaggioniI., CarboneD.P., FeoktistovI., and DikovM.M.Adenosine receptors in regulation of dendritic cell differentiation and function. Blood, 112, 1822, 2008.
31.
CiciarelloM., ZiniR., RossiL., SalvestriniV., FerrariD., ManfrediniR., and LemoliR.M.Extracellular purines promote the differentiation of human bone marrow-derived mesenchymal stem cells to the osteogenic and adipogenic lineages. Stem Cells Dev, 22, 1097, 2013.
32.
HuangS., and KamihiraM.Development of hybrid viral vectors for gene therapy. Biotechnol Adv, 31, 208, 2013.
33.
HadjantonakisA.K., PirityM., and NagyA.Cre recombinase mediated alterations of the mouse genome using embryonic stem cells. Methods Mol Biol, 461, 111, 2008.
34.
TangN., SongW.X., LuoJ., HaydonR.C., and HeT.C.Osteosarcoma development and stem cell differentiation. Clin Orthop Relat Res, 466, 2114, 2008.
35.
ShimizuT., IshikawaT., SugiharaE., KuninakaS., MiyamotoT., MabuchiY., MatsuzakiY., TsunodaT., MiyaF., MoriokaH., NakayamaR., KobayashiE., ToyamaY., KawaiA., IchikawaH., HasegawaT., OkadaS., ItoT., IkedaY., SudaT., and SayaH.c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis. Oncogene, 29, 5687, 2010.
36.
LinC.-Y., ChangY.-H., SungL.-Y., LuC.-H., ChenC.-L., LinS.-Y., LiK.-C., YenT.-C., LinK.-J., and HuY.-C.Long-term follow-up of massive segmental bone healing mediated by hybrid baculovirus-transduced ASCs: Focuses on bone remodeling and potential side effects. Tissue Eng Part A [Epub ahead of print]; DOI: 10.1089/ten.TEA.2013.0314, 2014.
37.
ReinkeS., GeisslerS., TaylorW.R., Schmidt-BleekK., JuelkeK., SchwachmeyerV., DahneM., HartwigT., AkyuzL., MeiselC., UnterwalderN., SinghN.B., ReinkeP., HaasN.P., VolkH.D., and DudaG.N.Terminally differentiated CD8(+) T cells negatively affect bone regeneration in humans. Sci Transl Med, 5, 177ra36, 2013.
38.
LiuY., WangL., KikuiriT., AkiyamaK., ChenC., XuX., YangR., ChenW.J., WangS., and ShiS.T.Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-γ and TNF-α. Nat Med, 17, 1594, 2012.
39.
DigheA.S., YangS., MadhuV., BalianG., and CuiQ.Interferon gamma and T cells inhibit osteogenesis induced by allogeneic mesenchymal stromal cells. J Orthop Res, 31, 227, 2013.
40.
LorenzoJ., HorowitzM., and ChoiY.Osteoimmunology: interactions of the bone and immune system. Endocr Rev, 29, 403, 2008.
41.
TobenD., SchroederI., El KhassawnaT., MehtaM., HoffmannJ.E., FrischJ.T., SchellH., LienauJ., SerraA., RadbruchA., and DudaG.N.Fracture healing is accelerated in the absence of the adaptive immune system. J Bone Miner Res, 26, 113, 2011.
42.
LuZ., WangG., DunstanC.R., ChenY., LuW.Y., DaviesB., and ZreiqatH.Activation and promotion of adipose stem cells by tumour necrosis factor-alpha preconditioning for bone regeneration. J Cell Physiol, 228, 1737, 2013.
43.
GerstenfeldL.C., ChoT.J., KonT., AizawaT., TsayA., FitchJ., BarnesG.L., GravesD.T., and EinhornT.A.Impaired fracture healing in the absence of TNF-alpha signaling: the role of TNF-alpha in endochondral cartilage resorption. J Bone Miner Res, 18, 1584, 2003.
44.
YamaguchiT., TakadaY., MaruyamaK., ShimodaK., AraiY., NangoN., KosakiN., TakaishiH., ToyamaY., and MatsuoK.Fra-1/AP-1 impairs inflammatory responses and chondrogenesis in fracture healing. J Bone Miner Res, 24, 2056, 2009.
45.
YangX., RicciardiB.F., Hernandez-SoriaA., ShiY., Pleshko CamachoN., and BostromM.P.Callus mineralization and maturation are delayed during fracture healing in interleukin-6 knockout mice. Bone, 41, 928, 2007.
46.
PhillipsJ.E., GersbachC.A., and GarciaA.J.Virus-based gene therapy strategies for bone regeneration. Biomaterials, 28, 211, 2007.
DonsanteA., MillerD.G., LiY., VoglerC., BruntE.M., RussellD.W., and SandsM.S.AAV vector integration sites in mouse hepatocellular carcinoma. Science, 317, 477, 2007.
49.
SchambachA., ZychlinskiD., EhrnstroemB., and BaumC.Biosafety features of lentiviral vectors. Hum Gene Ther, 24, 132, 2013.
50.
LuoW.Y., LinS.Y., LoK.W., LuC.H., HungC.L., ChenC.Y., ChangC.C., and HuY.C.Adaptive immune responses elicited by baculovirus and impacts on subsequent transgene expression in vivo. J Virol, 87, 4965, 2013.
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