Abstract
Lentiviral vectors (LV) are widely used to successfully transduce cells for research and clinical applications. This optimized LV infection protocol includes a nontoxic poloxamer-based adjuvant combined with antibody-retargeted lentiviral particles. The novel poloxamer P338 demonstrates superior characteristics for enhancing lentiviral transduction over the best-in-class polybrene-assisted transduction. Poloxamer P338 exhibited dual benefits of low toxicity and high efficiency of lentiviral gene delivery into a range of different primary cell cultures. One of the major advantages of P338 is its availability in pharma grade and applicability as cell culture medium additive in clinical protocols. Lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) can be produced to high titers and mediate high transduction efficiencies in vitro. For clinical applications the need for optimized transduction protocols, especially for transduction of primary T and stem cells, is high. The successful use of retronectin, the second lentivirus enhancer available as GMP material, requires the application of specific coating protocols not applicable in all processes, and results in the need of a relatively high multiplicity of infection (MOI) to achieve effective transduction efficiencies for hematopoietic cells (e.g., CD34+ hematopoietic stem cells). Cell specificity of lentiviral vectors was successfully increased by displaying different ratios of scFv-fused VSV-G glycoproteins on the viral envelope. The system has been validated with human CD30+ lymphoma cells, resulting in preferential gene delivery to CD30+ cells, which was increased fourfold in mixed cell cultures, by presenting scFv antibody fragments binding to respective surface markers. A combination of spinoculation and poloxamer-based chemical adjuvant increases the transduction of primary T-cells by greater than twofold. The combination of poloxamer-based and scFv-retargeted LVs increased transduction of CD30+ lymphoma cells more than tenfold, and has the potential to improve clinical protocols.
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References
Bukrinsky MI, Haggerty S, Dempsey MP et al (1993) A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365:666–669
Millington M, Arndt A, Boyd M et al (2009) Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells. PLoS One 4:e6461
Birmingham A, Anderson E, Sullivan K et al (2007) A protocol for designing siRNAs with high functionality and specificity. Nat Protoc 2:2068–2078
Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872
Anastasov N, Klier M, Koch I et al (2009) Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer. J Hematop 2:9–19
Anastasov N, Bonzheim I, Rudelius M et al (2010) C/EBPbeta expression in ALK-positive anaplastic large cell lymphomas is required for cell proliferation and is induced by the STAT3 signaling pathway. Haematologica 95:760–767
Wurm M, Schambach A, Lindemann D et al (2010) The influence of semen-derived enhancer of virus infection on the efficiency of retroviral gene transfer. J Gene Med 12:137–146
Burns JC, Friedmann T, Driever W et al (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A 90:8033–8037
Lin P, Correa D, Lin Y et al (2011) Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction. PLoS One 6:e23891
Ingrao D, Majdoul S, Seye AK et al (2014) Concurrent measures of fusion and transduction efficiency of primary CD34+ cells with human immunodeficiency virus 1-based lentiviral vectors reveal different effects of transduction enhancers. Hum Gene Ther Methods 25:48–56
Hesse J, Ebbesen P, Kristensen G (1978) Correlation between polyion effect on cell susceptibility to in vitro infection with murine C-type viruses and polyion effect on some membrane-related functions. Intervirology 9:173–183
Castro BA, Weiss CD, Wiviott LD et al (1988) Optimal conditions for recovery of the human immunodeficiency virus from peripheral blood mononuclear cells. J Clin Microbiol 26:2371–2376
Aubin RJ, Weinfeld M, Paterson MC (1988) Factors influencing efficiency and reproducibility of polybrene-assisted gene transfer. Somat Cell Mol Genet 14:155–167
Höfig I, Atkinson MJ, Mall S et al (2012) Poloxamer synperonic F108 improves cellular transduction with lentiviral vectors. J Gene Med 14:549–560
Lee RC, River LP, Pan FS et al (1992) Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo. Proc Natl Acad Sci U S A 89:4524–4528
Hannig J, Zhang D, Canaday DJ et al (2000) Surfactant sealing of membranes permeabilized by ionizing radiation. Radiat Res 154:171–177
Lu GW, Jun HW, Dzimianski MT et al (1995) Pharmacokinetic studies of methotrexate in plasma and synovial fluid following i.v. bolus and topical routes of administration in dogs. Pharm Res 12:1474–1477
Höfig I, Barth S, Salomon M et al (2014) Systematic improvement of lentivirus transduction protocols by antibody fragments fused to VSV-G as envelope glycoprotein. Biomaterials 35:4204–4212
Li Y, Drone C, Sat E et al (1993) Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains. J Virol 67:4070–4077
Lamb LS Jr, Bowersock J, Dasgupta A et al (2013) Engineered drug resistant gammadelta T cells kill glioblastoma cell lines during a chemotherapy challenge: a strategy for combining chemo- and immunotherapy. PLoS One 8:e51805
Strappe PM, Hampton DW, Cachon-Gonzalez B et al (2005) Delivery of a lentiviral vector in a Pluronic F127 gel to cells of the central nervous system. Eur J Pharm Biopharm 61:126–133
Waehler R, Russell SJ, Curiel DT (2007) Engineering targeted viral vectors for gene therapy. Nat Rev Genet 8:573–587
Guibinga GH, Hall FL, Gordon EM et al (2004) Ligand-modified vesicular stomatitis virus glycoprotein displays a temperature-sensitive intracellular trafficking and virus assembly phenotype. Mol Ther 9:76–84
Kameyama Y, Kawabe Y, Ito A et al (2008) Antibody-dependent gene transduction using gammaretroviral and lentiviral vectors pseudotyped with chimeric vesicular stomatitis virus glycoprotein. J Virol Methods 153:49–54
Padmashali RM, Andreadis ST (2011) Engineering fibrinogen-binding VSV-G envelope for spatially- and cell-controlled lentivirus delivery through fibrin hydrogels. Biomaterials 32:3330–3339
Kabanov A, Zhu J, Alakhov V (2005) Pluronic block copolymers for gene delivery. Adv Genet 53:231–261
Acknowledgments
This work was supported by the German Federal Ministry of Technology and Economics (ZIM-KOOP—KF2341801SB9). The authors would like to thank M. Salomon and S. Schrödel (Sirion Biotech GmbH, Martinsried, Germany) for technical support and J. Lintelmann (Cooperation Group Comprehensive Molecular Analytics, Helmholtz Center Munich, Germany) for performing mass spectrometric analyses. Published data was reprinted with kind permission from John Wiley & Sons Ltd. (Figs. 1 and 3) and Elsevier (Fig. 2).
DisclosuresC. Thirion is a founder and shareholder of Sirion Biotech GmbH. Other authors declare no conflict of interest.
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Anastasov, N., Höfig, I., Mall, S., Krackhardt, A.M., Thirion, C. (2016). Optimized Lentiviral Transduction Protocols by Use of a Poloxamer Enhancer, Spinoculation, and scFv-Antibody Fusions to VSV-G. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_4
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DOI: https://doi.org/10.1007/978-1-4939-3753-0_4
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