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J. Russick et al.
In fact, the very formulation of mRNA in TransIT® may be a reason for induction of a strong anti-FVIII immune response. TransIT® was initially conceived for in vitro and not in vivo gene transfection. As explained above, protein production following injection of TransIT®-formulated mRNA is not targeted to the liver or endothelial cells, which produce FVIII under physiologi- cal conditions. This is reminiscent of the early works on gene therapy for hemophilia wherein the use of promot- ers with poor specificity for hepatocytes was associated with the induction of neutralizing anti-FVIII or anti-fac- tor IX antibodies.47
Several lines of evidence suggest that the FVIII produced may itself be responsible for the sharp anti-FVIII immune response. As explained above, FVIII is a particularly immunogenic glycoprotein: e.g., a fusion protein between the light chain of FVIII and the first domain of hemagglu- tinin 1 (HA1) demonstrated greater anti-HA1 immuno- genicity upon intravenous injection to FVIII-deficient mice than the HA1 molecule alone.48 In contrast, the production of factor IX was induced in mice following administration of mRNA without report of a neutralizing immune response.18 Of note, a relationship between the dose of FVIII injected and the kinetics of detection of the anti-FVIII IgG response was reported in mice45 and in patients.49 This is particularly relevant in view of the fact that the amount of active FVIII produced over 72 h after one injection of FVIII-encoding mRNA was equivalent to 6-fold the
amount of rFVIII administered in a single injection. Furthermore, based on the poor specific activity of the endogenously produced FVIII, the total amount of FVIII molecules (active and inactive) probably exceeds 18-fold that of injected rFVIII. Further work will indicate whether targeting mRNA delivery to hepatocytes or to endothelial cells using improved lipid nanoparticle-based formulating agents,50 using mRNA encoding single chain or A2 variant stable FVIII39,40,41 and including miRNA target sequences to prevent off-target expression in hematopoietic cells51 are plausible strategies to improve the specific activity and reduce the immunogenicity of the endogenously pro- duced molecule.
Acknowledgment
We thank Dr Katalin Karikó (BioNTech RNA Pharmaceuticals, Mainz, Germany) for her constant support regarding this project and for providing the IVT mRNA. This study was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Sorbonne Université, and by grants from CSL-Behring (Paris, France), ANR (Exfiltrin project: ANR-18- CE17-0010-02) and CEFIPRA (IFC/7126/ Hemophilia/1353). JR was the recipient of a fellowship from Ministère de l'Enseignement Supérieur et de la Recherche. We also thank the staff from the Center of Histology, Cell Imaging and Flow Cytometry (CHIC) and Centre d'Expérimentation Fonctionnelle for assistance (Centre de Recherche des Cordeliers, Paris).
References
1. Mannucci PM, Tuddenham EG. The hemo- philias--from royal genes to gene therapy. N Engl J Med. 2001;344(23):1773-1779.
2. Oldenburg J, Mahlangu JN, Kim B, et al. Emicizumab prophylaxis in hemophilia A with inhibitors. N Engl J Med. 2017;377(9):809-818.
3. Chowdary P, Lethagen S, Friedrich U, et al. Safety and pharmacokinetics of anti-TFPI antibody (concizumab) in healthy volun- teers and patients with hemophilia: a ran- domized first human dose trial. J Thromb Haemost. 2015;13(5):743-754.
4. Pasi KJ, Rangarajan S, Georgiev P, et al. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N Engl J Med. 2017;377(9):819-828.
5. Polderdijk SG, Adams TE, Ivanciu L, et al. Design and characterization of an APC-spe- cific serpin for the treatment of hemophilia. Blood. 2017;129(1):105-113.
6. Nathwani AC, Davidoff AM, Tuddenham EGD. Advances in gene therapy for hemo- philia. Hum Gene Ther. 2017;28(11): 1004- 1012.
7. WolffJA,MaloneRW,WilliamsP,etal.Direct gene transfer into mouse muscle in vivo. Science. 1990;247(4949 Pt 1):1465-1468.
8. Heiser A, Coleman D, Dannull J, et al. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest. 2002;109(3):409-417.
9. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded
RNA and activation of NF-kappaB by Toll- like receptor 3. Nature. 2001;413(6857):732- 738.
10. Heil F, Hemmi H, Hochrein H, et al. Species- specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 2004;303(5663):1526-1529.
11. Hornung V, Ellegast J, Kim S, et al. 5'- Triphosphate RNA is the ligand for RIG-I. Science. 2006;314(5801):994-997.
12. Schlee M, Roth A, Hornung V, et al. Recognition of 5' triphosphate by RIG-I heli- case requires short blunt double-stranded RNA as contained in panhandle of negative- strand virus. Immunity. 2009;31(1):25-34.
13. Kariko K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modifi- cation and the evolutionary origin of RNA. Immunity. 2005;23(2):165-175.
14. Kariko K, Muramatsu H, Ludwig J, Weissman D. Generating the optimal mRNA for therapy: HPLC purification elim- inates immune activation and improves translation of nucleoside-modified, protein- encoding mRNA. Nucleic Acids Res. 2011;39(21):e142.
15. Kariko K, Muramatsu H, Keller JM, Weissman D. Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encod- ing erythropoietin. Mol Ther. 2012;20(5): 948-953.
16. Li B, Luo X, Deng B, et al. An orthogonal array optimization of lipid-like nanoparti- cles for mRNA delivery in vivo. Nano Lett. 2015;15(12):8099-8107.
17. Thess A, Grund S, Mui BL, et al. Sequence- engineered mRNA without chemical nucle-
oside modifications enables an effective pro- tein therapy in large animals. Mol Ther. 2015;23(9):1456-1464.
18. Ramaswamy S, Tonnu N, Tachikawa K, et al. Systemic delivery of factor IX messenger RNA for protein replacement therapy. Proc Natl Acad Sci U S A. 2017;114(10):E1941- E1950.
19. Pardi N, Hogan MJ, Pelc RS, et al. Zika virus protection by a single low-dose nucleoside- modified mRNA vaccination. Nature. 2017;543(7644):248-251.
20. Richner JM, Himansu S, Dowd KA, et al. Modified mRNA vaccines protect against zika virus infection. Cell. 2017;168(6):1114- 1125 e1110.
21. Su Z, Dannull J, Heiser A, et al. Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells. Cancer Res. 2003;63(9):2127-2133.
22. Kyte JA, Kvalheim G, Lislerud K, et al. T cell responses in melanoma patients after vacci- nation with tumor-mRNA transfected den- dritic cells. Cancer Immunol Immunother. 2007;56(5):659-675.
23. Healey JF, Barrow RT, Tamim HM, et al. Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII. Blood. 1998;92(10):3701-3709.
24. McIntosh J, Lenting PJ, Rosales C, et al. Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vec- tor encoding a novel human factor VIII vari- ant. Blood. 2013;121(17):3335-3344.
25. Ward NJ, Buckley SM, Waddington SN, et al. Codon optimization of human factor VIII cDNAs leads to high-level expression.
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