Restricted accessResearch articleFirst published online 2016-07
Sequential Adeno-Associated Viral Vector Serotype 9–Green Fluorescent Protein Gene Transfer Causes Massive Inflammation and Intense Immune Response in Rat Striatum
Green fluorescent protein (GFP) is a broadly used live cell reporter for gene transduction although side effects associated with GFP in gene transfer are reported. The present study was designed to systematically examine host responses, including inflammatory and immune responses, induced by persistent overexpression of the GFP gene mediated by adeno-associated viral vector serotype 9 (AAV9), and their effects on GFP gene transduction in rat striatum. Our results show that host responses against AAV9-GFP transduction, and GFP transgene expression in the striatum exhibited a temporal and dose-dependent pattern. Both muscular and striatal delivery of AAV9-GFP increased levels of inflammation and immune reactions against sequential AAV9-GFP transduction in the striatum, leading to reduced levels of GFP expression. We also observed that rat sera from sequential administrations of AAV9-GFP group had significantly higher levels of neutralizing antibody against AAV9 vectors when compared with the age-matched rats. As excessive GFP can trigger vigorous inflammation and intense immune response after GFP gene transduction, the use of GFP as a live cell marker protein should be deliberated, especially in repeated administration studies.
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References
1.
MarshallJ, MolloyR, MossGW, et al.The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function. Neuron, 1995; 14:211–215.
2.
BaensM, NoelsH, BroeckxV, et al.The dark side of EGFP: Defective polyubiquitination. PLoS One, 2006; 1:e54.
3.
GotoH, YangB, PetersenD, et al.Transduction of green fluorescent protein increased oxidative stress and enhanced sensitivity to cytotoxic drugs in neuroblastoma cell lines. Mol Cancer Ther, 2003; 2:911–917.
4.
GreenbaumL, RothmannC, LavieR, et al.Green fluorescent protein photobleaching: A model for protein damage by endogenous and exogenous singlet oxygen. Biol Chem, 2000; 381:1251–1258.
5.
ComleyLH, WishartTM, BaxterB, et al.Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: Implications for neurodegeneration research. PLoS One, 2011; 6:e17639.
6.
KleinRL, DaytonRD, LeidenheimerNJ, et al.Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins. Mol Ther, 2006; 13:517–527.
7.
BankiewiczKS, ForsayethJ, EberlingJL, et al.Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC. Mol Ther, 2006; 14:564–570.
8.
MandelRJ, ManfredssonFP, FoustKD, et al.Recombinant adeno-associated viral vectors as therapeutic agents to treat neurological disorders. Mol Ther, 2006; 13:463–483.
9.
CearleyCN, WolfeJH. Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain. Mol Ther, 2006; 13:528–537.
10.
FoustKD, NurreE, MontgomeryCL, et al.Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009; 27:59–65.
11.
KleinRL, DaytonRD, TatomJB, et al.AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: Effects of serotype, promoter and purification method. Mol Ther, 2008; 16:89–96.
XueYQ, MaBF, ZhaoLR, et al.AAV9-mediated erythropoietin gene delivery into the brain protects nigral dopaminergic neurons in a rat model of Parkinson's disease. Gene Ther, 2010; 17:83–94.
14.
BrockstedtDG, PodsakoffGM, FongL, et al.Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration. Clin Immunol, 1999; 92:67–75.
15.
CiesielskaA, HadaczekP, MittermeyerG, et al.Cerebral infusion of AAV9 vector-encoding non-self proteins can elicit cell-mediated immune responses. Mol Ther, 2013; 21:158–166.
16.
GeY, PowellS, Van RoeyM, et al.Factors influencing the development of an anti-factor IX (FIX) immune response following administration of adeno-associated virus-FIX. Blood, 2001; 97:3733–3737.
17.
MingozziF, MausMV, HuiDJ, et al.CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med, 2007; 13:419–422.
18.
PedenCS, BurgerC, MuzyczkaN, et al.Circulating anti-wild-type adeno-associated virus type 2 (AAV2) antibodies inhibit recombinant AAV2 (rAAV2)-mediated, but not rAAV5-mediated, gene transfer in the brain. J Virol, 2004; 78:6344–6359.
19.
PedenCS, ManfredssonFP, ReimsniderSK, et al.Striatal readministration of rAAV vectors reveals an immune response against AAV2 capsids that can be circumvented. Mol Ther, 2009; 17:524–537.
20.
PetryH, BrooksA, OrmeA, et al.Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice. Gene Ther, 2008; 15:54–60.
21.
RaptiK, Louis-JeuneV, KohlbrennerE, et al.Neutralizing antibodies against AAV serotypes 1, 2, 6, and 9 in sera of commonly used animal models. Mol Ther, 2012; 20:73–83.
22.
SanftnerLM, SuzukiBM, DoroudchiMM, et al.Striatal delivery of rAAV-hAADC to rats with preexisting immunity to AAV. Mol Ther, 2004; 9:403–409.
23.
XiaoW, ChirmuleN, SchnellMA, et al.Route of administration determines induction of T-cell-independent humoral responses to adeno-associated virus vectors. Mol Ther, 2000; 1:323–329.
24.
ZaissAK, MuruveDA. Immunity to adeno-associated virus vectors in animals and humans: A continued challenge. Gene Ther, 2008; 15:808–816.
25.
YangC, YangWH, ChenSS, et al.Pre-immunization with an intramuscular injection of AAV9-human erythropoietin vectors reduces the vector-mediated transduction following re-administration in rat brain. PLoS One, 2013; 8:e63876.
26.
ReimsniderS, ManfredssonFP, MuzyczkaN, et al.Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat. Mol Ther, 2007; 15:1504–1511.
27.
KleinRL, DaytonRD, DiaczynskyCG, et al.Pronounced microgliosis and neurodegeneration in aged rats after tau gene transfer. Neurobiol Aging, 2010; 31:2091–2102.
28.
DuanWM, WidnerH, BrundinP. Temporal pattern of host responses against intrastriatal grafts of syngeneic, allogeneic or xenogeneic embryonic neuronal tissue in rats. Exp Brain Res, 1995; 104:227–242.
29.
KottermanMA, YinL, StrazzeriJM, et al.Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates. Gene Ther, 2015; 22:116–126.
30.
ChirmuleN, XiaoW, TrunehA, et al.Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle. J Virol, 2000; 74:2420–2425.
31.
CodyCW, PrasherDC, WestlerWM, et al.Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry, 1993; 32:1212–1218.
32.
LiuHS, JanMS, ChouCK, et al.Is green fluorescent protein toxic to the living cells?. Biochem Biophys Res Commun, 1999; 260:712–717.
33.
KrestelHE, MihaljevicAL, HoffmanDA, et al.Neuronal co-expression of EGFP and beta-galactosidase in mice causes neuropathology and premature death. Neurobiol Dis, 2004; 17:310–318.
34.
WallaceLM, MoreoA, ClarkKR, et al.Dose-dependent toxicity of humanized Renilla reniformis GFP (hrGFP) limits its utility as a reporter gene in mouse muscle. Mol Ther Nucleic Acids, 2013; 2:e86.
35.
VillaP, BiginiP, MenniniT, et al.Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med, 2003; 198:971–975.
36.
XueYQ, ZhaoLR, GuoWP, et al.Intrastriatal administration of erythropoietin protects dopaminergic neurons and improves neurobehavioral outcome in a rat model of Parkinson's disease. Neuroscience, 2007; 146:1245–1258.
37.
SignoreAP, WengZ, HastingsT, et al.Erythropoietin protects against 6-hydroxydopamine-induced dopaminergic cell death. J Neurochem, 2006; 96:428–443.
38.
SirenAL, FratelliM, BrinesM, et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A, 2001; 98:4044–4049.
39.
LiuR, SuzukiA, GuoZ, et al.Intrinsic and extrinsic erythropoietin enhances neuroprotection against ischemia and reperfusion injury in vitro. J Neurochem, 2006; 96:1101–1110.
40.
DigicayliogluM, LiptonSA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature, 2001; 412:641–647.
41.
ManningWC, ZhouS, BlandMP, et al.Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors. Hum Gene Ther, 1998; 9:477–485.
42.
MaysLE, VandenbergheLH, XiaoR, et al.Adeno-associated virus capsid structure drives CD4-dependent CD8+ T cell response to vector encoded proteins. J Immunol, 2009; 182:6051–6060.
43.
VeronP, LeborgneC, MonteilhetV, et al.Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors. J Immunol, 2012; 188:6418–6424.
44.
LiC, HirschM, AsokanA, et al.Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo. J Virol, 2007; 81:7540–7547.
45.
LiH, MurphySL, Giles-DavisW, et al.Pre-existing AAV capsid-specific CD8+ T cells are unable to eliminate AAV-transduced hepatocytes. Mol Ther, 2007; 15:792–800.
46.
MastakovMY, BaerK, SymesCW, et al.Immunological aspects of recombinant adeno-associated virus delivery to the mammalian brain. J Virol, 2002; 76:8446–8454.
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