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Synthetic Messenger RNA and Cell Metabolism Modulation pp 127–138Cite as

Large Volume Flow Electroporation of mRNA: Clinical Scale Process

Part of the Methods in Molecular Biology book series (MIMB,volume 969)

Abstract

Genetic modification for enhancing cellular function has been continuously pursued for fighting diseases. Messenger RNA (mRNA) transfection is found to be a promising solution in modifying hematopoietic and immune cells for therapeutic purpose. We have developed a flow electroporation-based system for large volume electroporation of cells with various molecules, including mRNA. This allows robust and scalable mRNA transfection of primary cells of different origin. Here we describe transfection of chimeric antigen receptor (CAR) mRNA into NK cells to modulate the ability of NK cells to target tumor cells. High levels of CAR expression in NK cells can be maintained for 3–7 days post transfection. CD19-specific CAR mRNA transfected NK cells demonstrate targeted lysis of CD19-expressing tumor cells OP-1, primary B-CLL tumor cells, and autologous CD19+ B cells in in vitro assays with enhanced potency: >80% lysis at effector–target ratio of 1:1. This allows current good manufacturing practices (cGMP) and regulatory compliant manufacture of CAR mRNA transfected NK cells for clinical delivery.

Key words

  • mRNA transfection
  • NK cells
  • Flow electroporation
  • Large volume transfection
  • Scalability
  • Chimeric antigen receptor

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References

  1. Fischer A, Hacein-Bey-Abina S, Cavazzana-Calvo M (2011) Gene therapy for primary adaptive immune deficiencies. J Allergy Clin Immunol 127(6):1356–1359

    CrossRef  PubMed  CAS  Google Scholar 

  2. Thacker EE, Timares L, Matthews QL (2009) Strategies to overcome host immunity to adenovirus vectors in vaccine development. Expert Rev Vaccines 8(6):761–777

    CrossRef  PubMed  CAS  Google Scholar 

  3. Aaronson SA, Martin MA (1970) Transformation of human cells with different forms of SV40 DNA. Virology 42(4):848–856

    CrossRef  PubMed  CAS  Google Scholar 

  4. Graham FL, van der Eb AJ (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2):456–467

    CrossRef  PubMed  CAS  Google Scholar 

  5. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1(7):841–845

    PubMed  CAS  Google Scholar 

  6. Mac Gabhann F, Annex BH, Popel AS (2010) Gene therapy from the perspective of systems biology. Curr Opin Mol Ther 12(5):570–577

    PubMed  CAS  Google Scholar 

  7. Jensen MC, Popplewell L, Cooper LJ, DiGiusto D, Kalos M, Ostberg JR, Forman SJ (2010) Antitransgene rejection responses contribute to attenuated persistence of adoptively transferred CD20/CD19-specific chimeric antigen receptor redirected T cells in humans. Biol Blood Marrow Transplant 16(9):1245–1256

    CrossRef  PubMed  CAS  Google Scholar 

  8. Li LH, Biagi E, Allen C, Shivakumar R, Weiss JM, Feller S, Yvon E, Fratantoni JC, Liu LN (2006) Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells. Cancer Gene Ther 13(2):215–224

    CrossRef  PubMed  CAS  Google Scholar 

  9. Golzio M, Rols MP, Teissie J (2004) In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression. Methods 33(2):126–135

    CrossRef  PubMed  CAS  Google Scholar 

  10. Nishikawa M, Huang L (2001) Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther 12(8):861–870

    CrossRef  PubMed  CAS  Google Scholar 

  11. Hui SW, Li LH (2000) In vitro and ex vivo gene delivery to cells by electroporation. Methods Mol Med 37:157–171

    PubMed  CAS  Google Scholar 

  12. Xie TD, Sun L, Tsong TY (1990) Study of mechanisms of electric field-induced DNA transfection. I. DNA entry by surface binding and diffusion through membrane pores. Biophys J 58(1):13–19

    CrossRef  PubMed  CAS  Google Scholar 

  13. Zimmermann U (1986) Electrical breakdown, electropermeabilization and electrofusion. Rev Physiol Biochem Pharmacol 105:176–256

    PubMed  CAS  Google Scholar 

  14. Lai W, Chang CH, Farber DL (2003) Gene transfection and expression in resting and activated murine CD4 T cell subsets. J Immunol Methods 282(1–2):93–102

    CrossRef  PubMed  CAS  Google Scholar 

  15. Li LH, McCarthy P, Hui SW (2001) High-efficiency electrotransfection of human primary hematopoietic stem cells. FASEB J 15(3):586–588

    PubMed  CAS  Google Scholar 

  16. Van Tendeloo VF, Willems R, Ponsaerts P, Lenjou M, Nijs G, Vanhove M, Muylaert P, Van Cauwelaert P, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN (2000) High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery. Gene Ther 7(16):1431–1437

    CrossRef  PubMed  Google Scholar 

  17. Li LH, Sen A, Murphy SP, Jahreis GP, Fuji H, Hui SW (1999) Apoptosis induced by DNA uptake limits transfection efficiency. Exp Cell Res 253(2):541–550

    CrossRef  PubMed  CAS  Google Scholar 

  18. Van De Parre TJ, Martinet W, Schrijvers DM, Herman AG, De Meyer GR (2005) mRNA but not plasmid DNA is efficiently transfected in murine J774A.1 macrophages. Biochem Biophys Res Commun 327(1):356–360

    CrossRef  Google Scholar 

  19. Trompeter HI, Weinhold S, Thiel C, Wernet P, Uhrberg M (2003) Rapid and highly efficient gene transfer into natural killer cells by nucleofection. J Immunol Methods 274(1–2):245–256

    CrossRef  PubMed  CAS  Google Scholar 

  20. Ebert O, Finke S, Salahi A, Herrmann M, Trojaneck B, Lefterova P, Wagner E, Kircheis R, Huhn D, Schriever F, Schmidt-Wolf IG (1997) Lymphocyte apoptosis: induction by gene transfer techniques. Gene Ther 4(4):296–302

    CrossRef  PubMed  CAS  Google Scholar 

  21. Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, Chew A, Carroll RG, Scholler J, Levine BL, Albelda SM, June CH (2010) Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res 70(22):9053–9061

    CrossRef  PubMed  CAS  Google Scholar 

  22. Li L, Liu LN, Feller S, Allen C, Shivakumar R, Fratantoni J, Wolfraim LA, Fujisaki H, Campana D, Chopas N, Dzekunov S, Peshwa M (2010) Expression of chimeric antigen receptors in natural killer cells with a regulatory-compliant non-viral method. Cancer Gene Ther 17(3):147–154

    CrossRef  PubMed  CAS  Google Scholar 

  23. Rabinovich PM, Komarovskaya ME, Ye ZJ, Imai C, Campana D, Bahceci E, Weissman SM (2006) Synthetic messenger RNA as a tool for gene therapy. Hum Gene Ther 17(10):1027–1035

    CrossRef  PubMed  CAS  Google Scholar 

  24. Rabinovich PM, Komarovskaya ME, Wrzesinski SH, Alderman JL, Budak-Alpdogan T, Karpikov A, Guo H, Flavell RA, Cheung NK, Weissman SM, Bahceci E (2009) Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes. Hum Gene Ther 20(1):51–61

    CrossRef  PubMed  CAS  Google Scholar 

  25. Zhao Y, Zheng Z, Cohen CJ, Gattinoni L, Palmer DC, Restifo NP, Rosenberg SA, Morgan RA (2006) High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol Ther 13(1):151–159

    CrossRef  PubMed  CAS  Google Scholar 

  26. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7(5):618–630

    CrossRef  PubMed  CAS  Google Scholar 

  27. Yakubov E, Rechavi G, Rozenblatt S, Givol D (2010) Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophys Res Commun 394(1):189–193

    CrossRef  PubMed  CAS  Google Scholar 

  28. Pruitt SK, Boczkowski D, de Rosa N, Haley NR, Morse MA, Tyler DS, Dannull J, Nair S (2011) Enhancement of anti-tumor immunity through local modulation of CTLA-4 and GITR by dendritic cells. Eur J Immunol 41(12):3553–3563

    Google Scholar 

  29. Teufel R, Carralot JP, Scheel B, Probst J, Walter S, Jung G, Hoerr I, Rammensee HG, Pascolo S (2005) Human peripheral blood mononuclear cells transfected with messenger RNA stimulate antigen-specific cytotoxic T-lymphocytes in vitro. Cell Mol Life Sci 62(15):1755–1762

    CrossRef  PubMed  CAS  Google Scholar 

  30. Brentjens RJ, Riviere I, Park JH, Davila ML, Wang X, Stefanski J, Taylor C, Yeh R, Bartido S, Borquez-Ojeda O, Olszewska M, Bernal Y, Pegram H, Przybylowski M, Hollyman D, Usachenko Y, Pirraglia D, Hosey J, Santos E, Halton E, Maslak P, Scheinberg D, Jurcic J, Heaney M, Heller G, Frattini M, Sadelain M (2011) Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 118(18):4817–4828

    CrossRef  PubMed  CAS  Google Scholar 

  31. Hudecek M, Schmitt TM, Baskar S, Lupo-Stanghellini MT, Nishida T, Yamamoto TN, Bleakley M, Turtle CJ, Chang WC, Greisman HA, Wood B, Maloney DG, Jensen MC, Rader C, Riddell SR (2010) The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood 116(22):4532–4541

    CrossRef  PubMed  CAS  Google Scholar 

  32. Eshhar Z (2008) The T-body approach: redirecting T cells with antibody specificity. Handb Exp Pharmacol 181:329–342

    CrossRef  PubMed  CAS  Google Scholar 

  33. Imai C, Iwamoto S, Campana D (2005) Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 106(1):376–383

    CrossRef  PubMed  CAS  Google Scholar 

  34. Biagi E, Marin V, Giordano Attianese GM, Dander E, D’Amico G, Biondi A (2007) Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies. Haematologica 92(3):381–388

    CrossRef  PubMed  Google Scholar 

  35. Finney HM, Akbar AN, Lawson AD (2004) Activation of resting human primary T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain. J Immunol 172(1):104–113

    PubMed  CAS  Google Scholar 

  36. Witting SR, Li LH, Jasti A, Allen C, Cornetta K, Brady J, Shivakumar R, Peshwa MV (2012) Efficient large volume lentiviral vector production using flow electroporation. Hum Gene Ther 23(2):243–249

    CrossRef  PubMed  CAS  Google Scholar 

  37. Li LH, Shivakumar R, Feller S, Allen C, Weiss JM, Dzekunov S, Singh V, Holaday J, Fratantoni J, Liu LN (2002) Highly efficient, large volume flow electroporation. Technol Cancer Res Treat 1(5):341–350

    PubMed  CAS  Google Scholar 

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Acknowledgments

The authors acknowledge Dr. Dario Campana for the in-depth discussion and guidance in developing CAR mRNA technology. Thanks are also to Dr. James Brady for assistance with critical review of the manuscript.

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Authors and Affiliations

  1. MaxCyte Inc., Gaithersburg, MD, USA

    Linhong Li, Cornell Allen & Madhusudan V. Peshwa

  2. Max Cyte, Inc., Gaithersburg, MD, USA

    Rama Shivakumar

Authors
  1. Linhong Li
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  2. Cornell Allen
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  3. Rama Shivakumar
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  4. Madhusudan V. Peshwa
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Corresponding author

Correspondence to Madhusudan V. Peshwa .

Editor information

Editors and Affiliations

  1. The Anlyan Center, Department of Genetics, Yale School of Medicine, Cedar Street 300, New Haven, 06520-8005, Connecticut, USA

    Peter M. Rabinovich

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Li, L., Allen, C., Shivakumar, R., Peshwa, M.V. (2013). Large Volume Flow Electroporation of mRNA: Clinical Scale Process. In: Rabinovich, P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology, vol 969. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-260-5_9

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  • DOI: https://doi.org/10.1007/978-1-62703-260-5_9

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-259-9

  • Online ISBN: 978-1-62703-260-5

  • eBook Packages: Springer Protocols

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