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miRNA-mediated post-transcriptional silencing of transgenes leads to increased adeno-associated viral vector yield and targeting specificity

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Abstract

The production of high-titer recombinant adeno-associated virus (rAAV) vector is essential for treatment of genetic diseases affecting the retina and choroid, where anatomical constraints may limit injectable volumes. Problematically, cytotoxicity arising from overexpression of the transgene during vector production frequently leads to a reduction in vector yield. Herein, we evaluate the use of microRNA (miRNA)-mediated silencing to limit overexpression of cytotoxic transgenes during packaging as a method of increasing vector yield. We examined if post-transcriptional regulation of transgenes during packaging via miRNA technology would lead to increased rAAV yields. Our results demonstrate that silencing of cytotoxic transgenes during production resulted in up to a 22-fold increase in vector yield. The inclusion of organ-specific miRNA sequences improved biosafety by limiting off-target expression following systemic rAAV administration. The small size (22–23 bp) of the target site allows for the inclusion of multiple copies into the vector with minimal impact on coding capacity. Taken together, our results suggest that inclusion of miRNA target sites into the 3′-untranslated region of the AAV cassette allow for silencing of cytotoxic transgenes during vector production leading to improved vector yield, in addition to increasing targeting specificity without reliance on cell-specific promoters.

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References

  1. Day TP, Byrne LC, Schaffer DV, Flannery JG . Advances in AAV vector development for gene therapy in the retina. Adv Exp Med Biol 2014; 801: 687–693.

    Article  Google Scholar 

  2. Hammoudi N, Ishikawa K, Hajjar RJ . Adeno-associated virus-mediated gene therapy in cardiovascular disease. Curr Opin Cardiol 2015; 30: 228–234.

    Article  Google Scholar 

  3. Heldermon CD, Qin EY, Ohlemiller KK, Herzog ED, Brown JR, Vogler C et al. Disease correction by combined neonatal intracranial AAV and systemic lentiviral gene therapy in Sanfilippo Syndrome type B mice. Gene Ther 2013; 20: 913–921.

    Article  CAS  Google Scholar 

  4. Lehrke M, Lebherz C . AAV-mediated gene therapy for atherosclerosis. Curr Atheroscler Rep 2014; 16: 434.

    Article  Google Scholar 

  5. Ojala DS, Amara DP, Schaffer DV . Adeno-associated virus vectors and neurological gene therapy. Neuroscientist 2015; 21: 84–98.

    Article  Google Scholar 

  6. Santiago-Ortiz JL, Schaffer DV . Adeno-associated virus (AAV) vectors in cancer gene therapy. J Control Release 2016; 28: 287–301.

    Article  Google Scholar 

  7. Monahan PE, Sun J, Gui T, Hu G, Hannah WB, Wichlan DG et al. Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial. Hum Gene Ther 2015; 26: 69–81.

    Article  CAS  Google Scholar 

  8. Kaplitt MG, Feigin A, Tang C, Fitzsimons HL, Mattis P, Lawlor PA et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet 2007; 369: 2097–2105.

    Article  CAS  Google Scholar 

  9. Bakay RAEMDA Z, Tuszynski M, Potkin S, Bartus R, Bennett David . Analyses of a phase 1 clinical trial of adeno- associated virus-nerve growth factor (CERE-110) gene therapy in Alzheimer's disease. Clin Med 2007; 12: 240–207.

    Google Scholar 

  10. Janson C, McPhee S, Bilaniuk L, Haselgrove J, Testaiuti M, Freese A et al. Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain. Hum Gene Ther 2002; 13: 1391–1412.

    Article  CAS  Google Scholar 

  11. Cideciyan AV, Hauswirth WW, Aleman TS, Kaushal S, Schwartz SB, Boye SL et al. Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year. Hum Gene Ther 2009; 20: 999–1004.

    Article  CAS  Google Scholar 

  12. Cwerman-Thibault H, Augustin S, Ellouze S, Sahel JA, Corral-Debrinski M . Gene therapy for mitochondrial diseases: Leber Hereditary Optic Neuropathy as the first candidate for a clinical trial. C R Biol 2014; 337: 193–206.

    Article  Google Scholar 

  13. MacLaren RE, Groppe M, Barnard AR, Cottriall CL, Tolmachova T, Seymour L et al. Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial. Lancet 2014; 383: 1129–1137.

    Article  CAS  Google Scholar 

  14. Simonelli F, Maguire AM, Testa F, Pierce EA, Mingozzi F, Bennicelli JL et al. Gene therapy for Leber's congenital amaurosis is safe and effective through 1.5 years after vector administration. Mol Ther 2010; 18: 643–650.

    Article  CAS  Google Scholar 

  15. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342–347.

    Article  CAS  Google Scholar 

  16. Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 2011; 365: 2357–2365.

    Article  CAS  Google Scholar 

  17. Bowles DE, McPhee SW, Li C, Gray SJ, Samulski JJ, Camp AS et al. Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector. Mol Ther 2012; 20: 443–455.

    Article  CAS  Google Scholar 

  18. Ferreira V, Twisk J, Kwikkers K, Aronica E, Brisson D, Methot J et al. Immune responses to intramuscular administration of alipogene tiparvovec (AAV1-LPL(S447X)) in a phase II clinical trial of lipoprotein lipase deficiency gene therapy. Hum Gene Ther 2014; 25: 180–188.

    Article  CAS  Google Scholar 

  19. Jiang H, Pierce GF, Ozelo MC, de Paula EV, Vargas JA, Smith P et al. Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B. Mol Ther 2006; 14: 452–455.

    Article  CAS  Google Scholar 

  20. Mendell JR, Sahenk Z, Malik V, Gomez AM, Flanigan KM, Lowes LP et al. A phase 1/2a follistatin gene therapy trial for becker muscular dystrophy. Mol Ther 2015; 23: 192–201.

    Article  CAS  Google Scholar 

  21. Mietzsch M, Grasse S, Zurawski C, Weger S, Bennett A, Agbandje-McKenna M et al. OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1-12 vectors for gene therapy. Hum Gene Ther 2014; 25: 212–222.

    Article  CAS  Google Scholar 

  22. Mietzsch M, Casteleyn V, Weger S, Zolotukhin S, Heilbronn R . OneBac 2.0: Sf9 cell lines for production of AAV5 vectors with enhanced infectivity and minimal encapsidation of foreign DNA. Hum Gene Ther 2015; 26: 688–697.

    Article  CAS  Google Scholar 

  23. Strobel B, Klauser B, Hartig JS, Lamla T, Gantner F, Kreuz S . Riboswitch-mediated attenuation of transgene cytotoxicity increases adeno-associated virus vector yields in HEK-293 cells. Mol Ther 2015; 23: 1582–1591.

    Article  CAS  Google Scholar 

  24. Huntzinger E, Izaurralde E . Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 2011; 12: 99–110.

    Article  CAS  Google Scholar 

  25. Preusse M, Theis FJ, Mueller NS . miTALOS v2: analyzing tissue specific microRNA function. PLoS One 2016; 11: e0151771.

    Article  Google Scholar 

  26. Wienholds E, Plasterk RH . MicroRNA function in animal development. FEBS Lett 2005; 579: 5911–5922.

    Article  CAS  Google Scholar 

  27. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A et al. A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing. Cell 2007; 129: 1401–1414.

    Article  CAS  Google Scholar 

  28. Ludwig N, Leidinger P, Becker K, Backes C, Fehlmann T, Pallasch C et al. Distribution of miRNA expression across human tissues. Nucleic Acids Res 2016; 44: 3865–3877.

    Article  CAS  Google Scholar 

  29. Brown BD, Gentner B, Cantore A, Colleoni S, Amendola M, Zingale A et al. Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat Biotechnol 2007; 25: 1457–1467.

    Article  CAS  Google Scholar 

  30. Karali M, Manfredi A, Puppo A, Marrocco E, Gargiulo A, Allocca M et al. MicroRNA-restricted transgene expression in the retina. PLoS One 2011; 6: e22166.

    Article  CAS  Google Scholar 

  31. Xie J, Xie Q, Zhang H, Ameres SL, Hung J-H, Su Q et al. MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Mol Ther 2011; 19: 526–535.

    Article  CAS  Google Scholar 

  32. Zhong L, Li B, Mah CS, Govindasamy L, Agbandje-McKenna M, Cooper M et al. Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci USA 2008; 105: 7827–7832.

    Article  CAS  Google Scholar 

  33. Piedra J, Ontiveros M, Miravet S, Penalva C, Monfar M, Chillon M . Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors. Hum Gene Ther Methods 2015; 26: 35–42.

    Article  CAS  Google Scholar 

  34. Alekseenko IV, Snezhkov EV, Chernov IP, Pleshkan VV, Potapov VK, Sass AV et al. Therapeutic properties of a vector carrying the HSV thymidine kinase and GM-CSF genes and delivered as a complex with a cationic copolymer. J Transl Med 2015; 13: 78.

    Article  Google Scholar 

  35. Kim YH, Kim KT, Lee SJ, Hong SH, Moon JY, Yoon EK et al. Image-aided suicide gene therapy utilizing multifunctional hTERT-targeting adenovirus for clinical translation in hepatocellular carcinoma. Theranostics 2016; 6: 357–368.

    Article  CAS  Google Scholar 

  36. Kong H, Liu C, Zhu T, Huang Z, Yang L, Li Q . Effects of an adenoviral vector containing a suicide gene fusion on growth characteristics of breast cancer cells. Mol Med Rep 2014; 10: 3227–3232.

    Article  CAS  Google Scholar 

  37. Wu L, Zhou WB, Shen F, Liu W, Wu HW, Zhou SJ et al. Connexin32mediated antitumor effects of suicide gene therapy against hepatocellular carcinoma: In vitro and in vivo anticancer activity. Mol Med Rep 2016; 13: 3213–3219.

    Article  Google Scholar 

  38. Zhan H, Gilmour K, Chan L, Farzaneh F, McNicol AM, Xu JH et al. Production and first-in-man use of T cells engineered to express a HSVTK-CD34 sort-suicide gene. PLoS One 2013; 8: e77106.

    Article  CAS  Google Scholar 

  39. Oca-Cossio J, Kenyon L, Hao H, Moraes CT . Limitations of allotopic expression of mitochondrial genes in mammalian cells. Genetics 2003; 165: 707–720.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Petrs-Silva H, Dinculescu A, Li Q, Deng WT, Pang JJ, Min SH et al. Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina. Mol Ther 2011; 19: 293–301.

    Article  CAS  Google Scholar 

  41. Ryals RC, Boye SL, Dinculescu A, Hauswirth WW, Boye SE . Quantifying transduction efficiencies of unmodified and tyrosine capsid mutant AAV vectors in vitro using two ocular cell lines. Mol Vision 2011; 17: 1090–1102.

    CAS  Google Scholar 

  42. Markusic DM, Herzog RW, Aslanidi GV, Hoffman BE, Li B, Li M et al. High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines. Mol Ther 2010; 18: 2048–2056.

    Article  CAS  Google Scholar 

  43. Shemiakina II, Ermakova GV, Cranfill PJ, Baird MA, Evans RA, Souslova EA et al A monomeric red fluorescent protein with low cytotoxicity. Nat Commun 2012; 3: 1204.

    Article  CAS  Google Scholar 

  44. Georgi C, Buerger J, Hillen W, Berens C . Promoter strength driving TetR determines the regulatory properties of Tet-controlled expression systems. PLoS One 2012; 7: e41620.

    Article  CAS  Google Scholar 

  45. Baron U, Bujard H . Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol 2000; 327: 401–421.

    Article  CAS  Google Scholar 

  46. Bonnet C, El-Amraoui A . Usher syndrome (sensorineural deafness and retinitis pigmentosa): pathogenesis, molecular diagnosis and therapeutic approaches. Curr Opin Neurol 2012; 25: 42–49.

    Article  CAS  Google Scholar 

  47. Geisler A, Jungmann A, Kurreck J, Poller W, Katus HA, Vetter R et al. microRNA122-regulated transgene expression increases specificity of cardiac gene transfer upon intravenous delivery of AAV9 vectors. Gene Ther 2011; 18: 199–209.

    Article  CAS  Google Scholar 

  48. Tian W, Dong X, Liu X, Wang G, Dong Z, Shen W et al. High-throughput functional microRNAs profiling by recombinant AAV-based microRNA sensor arrays. PLoS One 2012; 7: e29551.

    Article  CAS  Google Scholar 

  49. Karali M, Peluso I, Gennarino VA, Bilio M, Verde R, Lago G et al miRNeye: a microRNA expression atlas of the mouse eye BMC Genomics 2010; 11: 715.

    Article  CAS  Google Scholar 

  50. Davies WL, Carvalho LS, Hunt DM . SPLICE: a technique for generating in vitro spliced coding sequences from genomic DNA. BioTechniques 2007; 43: 785–789.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank Dr Q Li and Dr A Verma for providing access to the fluorescent plate reader. Authors would also like to thank the Retinal Gene Therapy Vector Laboratory (University of Florida) for large-scale vector production. DML was funded through a Fulbright-Fight for Sight UK Research Scholarship (#1396) and is supported by intra-mural funding from the Medical College of Wisconsin. Additional funding was provided by NIH grant P30EY021721 and an unrestricted grant to the University of Florida department of Ophthalmology from Research to Prevent Blindness.

Author contributions

CAR, SLB, WWH and DML designed the study. CAR carried out all in vitro experiments and produced the figures. CAR and DML carried out in vivo experiments. CAR and DML prepared the manuscript with input from co-authors.

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Correspondence to D M Lipinski.

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WWH and the University of Florida have a financial interest in the use of AAV therapies, and own equity in a company (AGTC) that might, in the future, commercialize some aspects of this work. The remaining authors declare no conflict of interest.

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Supplementary Information accompanies this paper on Gene Therapy website

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Reid, C., Boye, S., Hauswirth, W. et al. miRNA-mediated post-transcriptional silencing of transgenes leads to increased adeno-associated viral vector yield and targeting specificity. Gene Ther 24, 462–469 (2017). https://doi.org/10.1038/gt.2017.50

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