Recombinant adeno-associated virus (AAV) represents an efficient system for neuronal transduction. However, a potential drawback of AAV is its restricted packaging capacity of approximately 5 kb. To bypass this limitation, a number of dual- and triple-vector strategies divide the transgene(s) between two or three AAVs. The success of these approaches relies directly on efficient cotransduction of the component AAVs. Although proof of concept for these stratagems has been demonstrated, the underlying cotransduction rate has not been analyzed quantitatively. In this study, cotransduction efficiencies in both retina and hippocampus have been investigated, using two reporter AAVs expressing either a green (GFP) or red (DsR) fluorescent protein. Transduction efficiencies were monitored via microscopy, flow cytometry, and quantitative PCR. After viral transduction with 1.5×109 viral particles of each of the reporter AAVs, approximately one-third of the retinal cells expressed one or both transgenes at levels detectable by native fluorescence. Notably, the majority of the remaining retinal cells were also transduced and expressed the reporters at lower levels, which were detectable only by immunolabeling. Flow cytometric analysis demonstrated cotransduction rates of up to 55% with the two reporter AAVs in retinal cells. Modifying the ratio of the two coadministered AAVs resulted in altered mRNA expression levels of the two reporter genes in cotransduced cell populations. The study suggests that codelivery of AAV is an efficient means of expanding the therapeutic application of AAV in neurons.
Get full access to this article
View all access options for this article.
References
1.
AlloccaM., DoriaM., PetrilloM.et al.2008. Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. J. Clin. Invest., 118:1955–1964.
2.
AuricchioA., KobingerG., AnandV.et al.2001. Exchange of surface proteins imparts on viral cellular specificity and transduction characteristics: The retina as a model. Hum. Mol. Genet., 10:3075–3081.
3.
BainbridgeJ.W., SmithA.J., BarkerS.S.et al.2008. Effect of gene therapy on visual function in Leber's congenital amaurosis. N. Engl. J. Med., 22,358:2231–2239.
4.
BennicelliJ., WrightJ.F., KomaromyA.et al.2008. Reversal of blindness in animal models of Leber congenital amaurosis using optimized AAV2-mediated gene transfer. Mol. Ther., 16:458–465.
5.
BuchP.K., MacLarenR.E., DuránY.et al.2006. In contrast to AAV-mediated Cntf expression, AAV-mediated Gdnf expression enhances gene replacement therapy in rodent models of retinal degeneration. Mol. Ther., 14:700–709.
6.
BurgerC., GorbatyukO.S., VelardoM.J.et al.2004. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther., 10:302–317.
7.
BusskampV., DuebelJ., BalyaD.et al.2010. Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa. Science, 329:413–417.
8.
CaporaleN., KolstadK.D., LeeT.et al.2011. LiGluR restores visual responses in rodent models of inherited blindness. Mol. Ther., 19:1212–1219.
9.
CepkoC.2010. Neuroscience: Seeing the light of day. Science, 329:403–404.
10.
ChaddertonN., Millington-WardS., PalfiA.et al.2009. Improved retinal function in a mouse model of dominant retinitis pigmentosa following AAV-delivered gene therapy. Mol. Ther., 17:593–599.
11.
ChristineC.W., StarrP.A., LarsonP.S.et al.2009. Safety and tolerability of putaminal AADC gene therapy for Parkinson disease. Neurology, 73:1662–1669.
12.
DongB., NakaiH., XiaoW.2010. Characterization of genome integrity for oversized recombinant AAV vector. Mol. Ther., 18:87–92.
13.
DongJ.Y., FanP.D., FrizzellR.A.1996. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum. Gene Ther., 7:2101–2112.
14.
DoonanF., DonovanM., CotterT.G.2005. Activation of multiple pathways during photoreceptor apoptosis in the rd mouse. Invest. Ophthalmol. Vis. Sci., 46:3530–3538.
15.
DuanD., YueY., YanZ., EngelhardtJ.F.2000. A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation. Nat. Med., 6:595–598.
16.
FanD.S., OgawaM., FujimotoK.I.et al.1998. 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., 9:2527–2535.
GaoG., VandenbergheL.H., WilsonJ.M.2005. New recombinant serotypes of AAV vectors. Curr. Gene Ther., 5:285–297.
19.
GhoshA., YueY., DuanD.2006. Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle. J. Gene Med., 8:298–305.
20.
GhoshA., YueY., ShinJ.H., DuanD.2009. Systemic trans-splicing adeno-associated viral delivery efficiently transduces the heart of adult mdx mouse, a model for Duchenne muscular dystrophy. Hum. Gene Ther., 20:1319–1328.
21.
HalbertC.L., AllenJ.M., MillerA.D.2002. Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene. Nat. Biotechnol., 20:697–701.
22.
HamelC.P.2007. Cone rod dystrophies. Orphanet J. Rare Dis., 2:7.
HauswirthW.W., AlemanT.S., KaushalS.et al.2008. Treatment of Leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: Short-term results of a phase I trial. Hum. Gene Ther., 19:979–990.
25.
JacobsonS.G., AclandG.M., AguirreG.D.et al.2006. Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection. Mol. Ther., 13:1074–1084.
26.
JeonC.J., StrettoiE., MaslandR.H.1998. The major cell populations of the mouse retina. J. Neurosci., 18:8936–8946.
KimD.H., RossiJ.J.2003. Coupling of RNAi-mediated target downregulation with gene replacement. Antisense Nucleic Acid Drug Dev., 13:151–155.
29.
KomeimaK., RogersB.S., CampochiaroP.A.2007. Antioxidants slow photoreceptor cell death in mouse models of retinitis pigmentosa. J. Cell Physiol., 213:809–815.
30.
KuboderaT., YamadaH., AnzaiM.et al.2011. In vivo application of an RNAi strategy for the selective suppression of a mutant allele. Hum. Gene Ther., 22:27–34.
31.
LagaliP.S., BalyaD., AwatramaniG.B.et al.2008. Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat. Neurosci., 11:667–675.
32.
LaiY., YueY., DuanD.2010. Evidence for the failure of adeno-associated virus serotype 5 to package a viral genome ≥8.2 kb. Mol. Ther., 18:75–79.
33.
LiW., AsokanA., WuZ.et al.2008. Engineering and selection of shuffled AAV genomes: A new strategy for producing targeted biological nanoparticles. Mol. Ther., 16:1252–1260.
34.
LiX.G., OkadaT., KoderaM.et al.2006. Viral-mediated temporally controlled dopamine production in a rat model of Parkinson disease. Mol. Ther., 13:160–166.
35.
LiY., TaoW., LuoL.et al.2010. CNTF induces regeneration of cone outer segments in a rat model of retinal degeneration. PLoS One, 5:e9495.
36.
MacLachlanT.K., LukasonM., CollinsM.et al.2011. Preclinical safety evaluation of AAV2-sFLT01: A gene therapy for age-related macular degeneration. Mol. Ther., 19:326–334.
37.
MaguireA.M., SimonelliF., PierceE.A.et al.2008. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N. Engl. J. Med., 358:2240–2248.
MandelR.J.2010. CERE-110, an adeno-associated virus-based gene delivery vector expressing human nerve growth factor for the treatment of Alzheimer's disease. Curr. Opin. Mol. Ther., 12:240–247.
40.
MaoH., GorbatyukM.S., RossmillerB.et al.2012. Long term rescue of retinal structure and function in P23H RHO transgenic mice by rhodopsin RNA replacement with a single AAV vector. Hum. Gene Ther., 23:356–366.
41.
MarksW.J.Jr., OstremJ.L., VerhagenL.et al.2008. Safety and tolerability of intraputaminal delivery of CERE-120 (adeno-associated virus serotype 2-neurturin) to patients with idiopathic Parkinson's disease: An open-label, phase I trial. Lancet Neurol., 7:400–408.
42.
McCartyD.M.2008. Self-complementary AAV vectors: Advances and applications. Mol. Ther., 16:1648–1656.
43.
McKeeA.G., LoscherJ.S., O'SullivanN.C.et al.2010. AAV-mediated chronic over-expression of SNAP-25 in adult rat dorsal hippocampus impairs memory-associated synaptic plasticity. J. Neurochem., 112:991–1004
44.
Millington-WardS., O'NeillB., TuohyG.et al.1997. Stratagems in vitro for gene therapies directed to dominant mutations. Hum. Mol. Genet., 6:1415–1426.
45.
Millington-WardS., ChaddertonN., O'ReillyM.et al.2011. Suppression and replacement gene therapy for autosomal dominant disease in a murine model of dominant retinitis pigmentosa. Mol. Ther., 19:642–649.
46.
MonahanP.E., LothropC.D., SunJ.et al.2010. Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: A strategy for broad clinical application. Mol. Ther., 18:1907–1916.
47.
MuramatsuS., FujimotoK., IkeguchiK.et al.2002. Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes. Hum. Gene Ther., 13:345–354.
48.
MusarellaM.A.2001. Molecular genetics of macular degeneration. Doc. Ophthalmol., 102:165–177.
49.
O'ReillyM., PalfiA., ChaddertonN.et al.2007. RNA interference-mediated suppression and replacement of human rhodopsin in vivo. Am. J. Hum. Genet., 81:127–135.
50.
PalfiA., AderM., KiangA.S.et al.2006. RNAi-based suppression and replacement of rds-peripherin in retinal organotypic culture. Hum. Mutat., 27:260–268.
51.
PalfiA., Millington-WardS., ChaddertonN.et al.2010. AAV-mediated rhodopsin replacement provides therapeutic benefit in mice with a targeted disruption of the rhodopsin gene. Hum. Gene Ther., 21:311–323.
52.
PawlykB.S., SmithA.J., BuchP.K.et al.2005. Gene replacement therapy rescues photoreceptor degeneration in a murine model of Leber congenital amaurosis lacking RPGRIP. Invest. Ophthalmol. Vis. Sci., 46:3039–3045.
53.
ProvostN., Le MeurG., WeberM.et al.2005. Biodistribution of rAAV vectors following intraocular administration: Evidence for the presence and persistence of vector DNA in the optic nerve and in the brain. Mol. Ther., 11:275–283.
54.
ReichS.J., AuricchioA., HildingerM.et al.2003. Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy. Hum. Gene Ther., 14:37–44.
55.
StiegerK., ColleM.A., DubreilL.et al.2008. Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain. Mol. Ther., 16:916–923.
56.
StiegerK., SchroederJ., ProvostN.et al.2009. Detection of intact rAAV particles up to 6 years after successful gene transfer in the retina of dogs and primates. Mol. Ther., 17:516–523.
57.
WuZ., YangH., ColosiP.2010. Effect of genome size on AAV vector packaging. Mol. Ther., 18:80–86.
58.
YanD., LiuX.Z.2010. Genetics and pathological mechanisms of Usher syndrome. J. Hum. Genet., 55:327–335.
59.
YanZ., ZhangY., DuanD., EngelhardtJ.F.2000. Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc. Natl. Acad. Sci. U.S.A., 97:6716–6721.
60.
YangG.S., SchmidtM., YanZ.et al.2002. Virus-mediated transduction of murine retina with adeno-associated virus: Effects of viral capsid and genome size. J. Virol., 76:7651–7660.
61.
YangY., Mohand-SaidS., DananA.et al.2009. Functional cone rescue by RdCVF protein in a dominant model of retinitis pigmentosa. Mol. Ther., 17:787–795.
62.
ZhongL., LiB., MahC.S.et al.2008. Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc. Natl. Acad. Sci. U.S.A., 105:7827–7832.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
