Abstract
Recombinant adeno-associated viruses (rAAVs) are promising vectors for gene therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantage of rAAVs is their limited capacity to package foreign DNA (⩽5 kb). Often, co-expression of two or more genes from a single viral vector is desirable to achieve maximal therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translation can be relatively inefficient when compared with cap-dependent translation. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). The 2A peptide mediates the primary cis-‘cleavage’ of the FMDV polyprotein in a cascade of processing events that ultimately generate the mature FMDV proteins. We have generated several different rAAV genomes in which two coding regions are fused in-frame via the FMDV 2A sequence. We show that FMDV 2A efficiently mediates the generation of the expected cleavage products from the artificial fusion proteins in cells. Furthermore, we find that both EGFP and α- synuclein are expressed at substantially higher levels from 2A vectors than from the corresponding IRES-based vectors, while SOD-1 is expressed at comparable or slightly higher levels. Finally, we demonstrate for the first time, that the 2A sequence results in effective bicistronic gene expression in vivo after injection of 2A-dependent rAAVs into the rat substantia nigra. We conclude that 2A-containing rAAVs may represent an attractive alternative to IRES-dependent vectors for ex vivo and in vivo gene expression and gene therapy.
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References
Kaplitt MG et al. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain Nat Genet 1994 8: 148–154
McCown TJ et al. Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector Brain Res 1996 713: 99–107
Xiao X, Li J, Samulski RJ . Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector J Virol 1996 70: 8098–8108
Jooss K, Yang Y, Fisher KJ, Wilson JM . Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers J Virol 1998 72: 4212–4223
Snyder RO . Adeno-associated virus-mediated gene delivery J Gene Med 1999 1: 166–175
Bennett J et al. Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina Proc Natl Acad Sci USA 1999 96: 9920–9925
During MJ et al. An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy Science 2000 287: 1453–1460
Fisher KJ et al. Recombinant adeno-associated virus for muscle directed gene therapy Nat Med 1997 3: 306–312
Glatzel M et al. Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system Proc Natl Acad Sci USA 2000 97: 442–447
Herzog RW et al. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector Nat Med 1999 5: 56–63
Lewin AS et al. Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa (published erratum appears in Nat Med 1998; 4: 1081) Nat Med 1998 4: 967–971
Peel AL et al. Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters Gene Therapy 1997 4: 16–24
Snyder RO et al. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors Nat Med 1999 5: 64–70
Szczypka MS et al. Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice Neuron 1999 22: 167–178
Girod A et al. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2 Nat Med 1999 5: 1052–1056
Rabinowitz JE, Xiao W, Samulski RJ . Insertional mutagenesis of AAV2 capsid and the production of recombinant virus Virology 1999 265: 274–285
Wu P et al. Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism J Virol 2000 74: 8635–8647
Davidson BL et al. Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system Proc Natl Acad Sci USA 2000 97: 3428–3432
Zabner J et al. Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer J Virol 2000 74: 3852–3858
Dong JY, Fan PD, Frizzell RA . Quantitative analysis of the packaging capacity of recombinant adeno-associated virus Hum Gene Ther 1996 7: 2101–2112
Recchia A et al. Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector Proc Natl Acad Sci USA 1999 96: 2615–2620
Costantini LC et al. Gene transfer to the nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors Hum Gene Ther 1999 10: 2481–2494
Nakai H, Storm TA, Kay MA . Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors Nat Biotechnol 2000 18: 527–532
Klein RL, Lewis MH, Muzyczka N, Meyer EM . Prevention of 6-hydroxydopamine-induced rotational behavior by BDNF somatic gene transfer Brain Res 1999 847: 314–320
Levenson VV, Transue ED, Roninson IB . Internal ribosomal entry site-containing retroviral vectors with green fluorescent protein and drug resistance markers Hum Gene Ther 1998 9: 1233–1236
Martinez-Salas E . Internal ribosome entry site biology and its use in expression vectors Curr Opin Biotechnol 1999 10: 458–464
Borman AM, Le Mercier P, Girard M, Kean KM . Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins Nucleic Acids Res 1997 25: 925–932
Roberts LO, Seamons RA, Belsham GJ . Recognition of picornavirus internal ribosome entry sites within cells; influence of cellular and viral proteins RNA 1998 4: 520–529
Harries M et al. Comparison of bicistronic retroviral vectors containing internal ribosome entry sites (IRES) using expression of human interleukin-12 (IL-12) as a readout J Gene Med 2000 2: 243–249
Mizuguchi H et al. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector Mol Ther 2000 1: 376–382
Zhou Y, Aran J, Gottesman MM, Pastan I . Co-expression of human adenosine deaminase and multidrug resistance using a bicistronic retroviral vector Hum Gene Ther 1998 9: 287–293
Palmenberg AC . Proteolytic processing of picornaviral polyprotein Annu Rev Microbiol 1990 44: 603–623
Donnelly ML et al. The cleavage activities of aphthovirus and cardiovirus 2A proteins J Gen Virol 1997 78: 13–21
Ryan MD, King AM, Thomas GP . Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence J Gen Virol 1991 72: 2727–2732
Ryan MD, Drew J . Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein EMBO J 1994 13: 928–933
Gopinath K et al. Engineering cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants Virology 2000 267: 159–173
Halpin C et al. Self-processing 2A-polyproteins – a system for co-ordinate expression of multiple proteins in transgenic plants Plant J 1999 17: 453–459
Chaplin PJ et al. Production of interleukin-12 as a self-processing 2A polypeptide J Interfer Cytok Res 1999 19: 235–241
Percy N, Barclay WS, Garcia-Sastre A, Palese P . Expression of a foreign protein by influenza A virus J Virol 1994 68: 4486–4492
de Felipe P et al. Use of the 2A sequence from foot-and-mouth disease virus in the generation of retroviral vectors for gene therapy Gene Therapy 1999 6: 198–208
De Felipe P, Izquierdo M . Tricistronic and tetracistronic retroviral vectors for gene transfer Hum Gene Ther 2000 11: 1921–1931
Palmenberg AC et al. Proteolytic processing of the cardioviral P2 region: primary 2A/2B cleavage in clone-derived precursors Virology 1992 190: 754–762
Games D et al. Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein Nature 1995 373: 523–527
Masliah E et al. Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders Science 2000 287: 1265–1269
Fan DS et al. Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors Hum Gene Ther 1998 9: 2527–2535
Ryan MD et al. A model for nonstoichiometric, cotranslational protein scission in eukaryotic ribosomes Bioorg Chem 1999 27: 55–79
Mattion NM, Harnish EC, Crowley JC, Reilly PA . Foot-and-mouth disease virus 2A protease mediates cleavage in attenuated Sabin 3 poliovirus vectors engineered for delivery of foreign antigens J Virol 1996 70: 8124–8127
Ypma-Wong MF, Filman DJ, Hogle JM, Semler BL . Structural domains of the poliovirus polyprotein are major determinants for proteolytic cleavage at Gln-Gly pairs J Biol Chem 1988 263: 17846–17856
Chen SH et al. Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival Cancer Res 1996 56: 3758–3762
Moriuchi S et al. Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase Cancer Res 1998 58: 5731–5737
Pizzato M et al. Production and characterization of a bicistronic Moloney-based retroviral vector expressing human interleukin 2 and herpes simplex virus thymidine kinase for gene therapy of cancer Gene Therapy 1998 5: 1003–1007
Paterna JC et al. Influence of promoter and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in the rat brain Gene Therapy 2000 7: 1304–1311
Badorff C et al. Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy Nat Med 1999 5: 320–326
Kirchweger R et al. Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma J Virol 1994 68: 5677–5684
Lamphear BJ, Kirchweger R, Skern T, Rhoads RE . Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation J Biol Chem 1995 270: 21975–21983
Piccone ME et al. Identification of the active-site residues of the L proteinase of foot-and-mouth disease virus J Virol 1995 69: 4950–4956
Lamphear BJ et al. Mapping the cleavage site in protein synthesis initiation factor eIF-4 gamma of the 2A proteases from human Coxsackievirus and rhinovirus J Biol Chem 1993 268: 19200–19203
Hemmi S et al. The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures Hum Gene Ther 1998 9: 2363–2373
Donello JE, Loeb JE, Hope TJ . Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element J Virol 1998 72: 5085–5092
Zolotukhin S et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield Gene Therapy 1999 6: 973–985
Snyder RO, Xiao X, Samulski RJ . Production of recombinant adeno-associated viral vectors. In: Dracopoli NC et al (eds) Current Protocols in Human Genetics John Wiley: New York 1997 12.1.1.–12.1.20
Acknowledgements
We thank Martin Ryan for plasmid pTG395, Thomas Bächi and Zhizhong Dong for help with confocal microscopy and Fritz Ochsenbein for preparing figures. We are also grateful to Lee Martin for helpful discussion and critical reading of the manuscript. This work was supported by grants from the Swiss National Science Foundation to HB.
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Furler, S., Paterna, JC., Weibel, M. et al. Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons. Gene Ther 8, 864–873 (2001). https://doi.org/10.1038/sj.gt.3301469
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DOI: https://doi.org/10.1038/sj.gt.3301469