|Molecular Vision 2001;
Received 5 October 2000 | Accepted 19 February 2001 | Published 23 February 2001
Epitope-tagged recombinant AAV vectors for expressing neurturin and its receptor in retinal cells
Michael J. Neal,1
Stephen E. Jones1
1Retinitis Pigmentosa Research Unit, The Rayne Institute, Division of Pharmacology and Therapeutics, Guy's, King's and St Thomas' School of Biomedical Sciences, King's College London, St Thomas' Hospital, London, UK; 2Division of Laboratory of Medicine, Department of Pathology and Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO
Correspondence to: Catherine Jomary, Retinitis Pigmentosa Research Unit, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK; Phone: (44) 020 7928 9292; FAX: (44) 020 7922 8223; email: email@example.com
Purpose: Neurturin (NTN) is a potent neuronal survival factor in the central and peripheral nervous systems. We previously described altered expression of mRNAs for NTN and one of its receptor components, GFRa-2 in degenerative retinas of rd/rd mice. Towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (rAAV) constructs for expression of NTN and GFRa-2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mice.
Methods: The rAAV constructs incorporated epitope tags to facilitate discrimination between transgenic and endogenous expression. Expression of murine NTN was driven by a CMV promoter and a partial murine opsin promoter was used to drive expression of human GFRa-2. rAAV preparations were used to infect mouse retinal cell cultures and for intraocular injection of predegenerative rd/rd mice. Endogenous and transgene expression was analyzed by immunofluorescence. Photoreceptor function in treated mice was assessed by electroretinography.
Results: Both vectors delivered and expressed their transgenes in vitro and in vivo. Differential targeting was achieved in vivo through the use of alternative promoters. Under the conditions examined, no functional rescue of rd photoreceptors was observed.
Conclusions: Therapeutic treatment of the rd model of retinal degeneration does not appear to be effected by simple modulation of the expression of NTN or GFRa-2, and may therefore depend on additional synergistic factors. Our AAV constructs will facilitate the development of combinatorial approaches to the treatment of central and peripheral neurodegenerations.
Neurotrophic factors are molecules that promote survival, differentiation, and maintenance of neurons. Neurturin (NTN) is one of the four known members of the neurotrophic factor (GDNF) family derived from glial cell lines . It promotes the survival of cultured rat superior sympathetic, dorsal root and nodose ganglion neurons, midbrain dopaminergic neurons, and enteric neurons. NTN signals via multicomponent receptors that consist of the Ret receptor tyrosine kinase plus a member of a family of structurally related glycosyl-phosphatidylinositol (GPI)-linked receptors, GFRa-2 [1,2]. Recent studies have shown that NTN is the major physiologic ligand for GFRa-2 .
We previously reported a discrepant expression between NTN and its GFRa-2 receptor component in the rd mouse model of retinal degeneration, suggesting that dysregulation of the NTN neurotrophic function is associated with photoreceptor cell death . During the progress of degeneration in this model, there is upregulation of NTN mRNA expression (also NTN protein; data not shown) but GFRa-2 mRNA levels remain lower than in age-matched control retinas. We hypothesized that increased NTN expression is a survival-promoting response of the retina to the onset of degeneration, but that its potential neurotrophic effect on photoreceptors is constrained by the persistently low GFRa-2 levels in rd retinas. Alternatively, since NTN also signals through the GDNF receptor (GFRa-1) but via a low-affinity interaction , and GFRa-1 levels are similar in rd and control retinas (data not shown), it is possible that increased NTN is limited in its efficacy by failure to activate sufficient survival-promoting pathways through the GFRa-1 receptors. In view of these possibilities, and of the ability of exogenously-supplied NTN to rescue degenerative dopaminergic neurons , we have investigated delivery of NTN and GFRa-2 genes to murine retinal cells in vitro and in vivo, using the adeno-associated virus (AAV) vector, an effective vehicle for gene transfer to neurons in the brain and retina [4-6]. Further, to enable us to discriminate between the two transgenes and their counterpart endogenous genes, we have employed epitope tag systems in conjunction with specific antibodies.
AAV-vector construction, preparation, and delivery
The cytomegalovirus (CMV) promoter-murine NTN coding sequence of pJDM1923  was amplified by PCR, inserted into the C-terminal tag vector pcDNA3HisV5 (Invitrogen, Leek, The Netherlands), and transferred to the AAV vector, pTR (Figure 1A). A CMV-GFRa-2 plasmid with the influenza protein hemagglutinin (HA) tag inserted between amino acids 32 and 33 was adapted to excise the coding sequence and replace the PDE-b cDNA in AAV plasmid pTOP8, downstream of a 1.4 kb fragment of the proximal murine opsin promoter  (Figure 1B). Recombinant AAV virions were produced using standard protocols , purified by double cesium chloride density gradient centrifugation, dialysed, and the viral titer determined by infectious center assay . Titer was 2.6x109 infectious units/ml for AAV.NTN, and 4x107 infectious units/ml for AAV.GFRa-2. Helper adenovirus was detectable by immunocytochemistry using anti-adenoviral antibody (Chemicon International, Harrow, UK) on infected 293 cells (ECACC, Salisbury, UK) only in purified AAV.NTN, at a titer of 90 infectious units/ml. For in vitro studies, mouse retinal cell cultures (3.3-6.6x105 cells/ml) were infected with 2-5 ml of each rAAV preparation per 0.3 ml tissue culture medium . Intraocular injection of a maximum of 2 ml of rAAV preparations was performed on the left eye only of 7-8 day-old rd mice as previously described . Right eyes served as untreated controls. All animal procedures were carried out in accordance with the requirements of a Home Office License, and in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Retinal cell cultures were maintained for 48 h at 37 °C post-inoculation, fixed in 4% paraformaldehyde, and processed for immunocytochemistry using various combinations of a polyclonal goat anti-human NTN antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), a polyclonal goat anti-human GFRa-2 antibody (1:200; Santa Cruz), a monoclonal anti-V5 antibody (1:500; Invitrogen), and a monoclonal anti-HA protein (1:500; Santa Cruz). The antibodies were visualized as appropriate using a rabbit anti-goat conjugated to fluorescein (Sigma, Poole, Dorset UK), and a rabbit anti-mouse IgG conjugated to rhodamine (Sigma), at concentrations according to manufacturer's recommendations. Mice were killed at 10-11 days post-injection, the eyes enucleated and fixed in fresh 4% paraformaldehyde in 0.1 M phosphate buffer overnight, embedded and frozen . Cryostat sections (10 mm) were processed for immunocytochemistry as described for in vitro studies.
Mice were dark adapted for at least 12 h prior to electroretinographic (ERG) testing as described previously [12-14]. Briefly, mice were anesthetized with 80 mg/kg pentobarbital injected intraperitoneally, the full-field ERGs were elicited with a 10 ms flash of white light (4.6 log footlamberts, 1 footlambert=3.4 cd/m2) presented from a Ganzfeld dome, and recorded using commercially available equipment (EpTech ERG system, EpTech, Newark, Notts, UK).
Results & Discussion
Recombinant AAV (rAAV) vectors originate from nonpathogenic, single-stranded DNA viruses capable of infecting both dividing and non-dividing cells. Present vectors minimally comprise only the two inverted terminal repeat (ITR) sequences necessary for packaging of inserted sequences, and the vector is therefore devoid of any viral coding sequences, reducing the risk of undesirable host immune responses. Several reports have demonstrated the ability of rAAVs to mediate reporter gene transfer to the retina in several species [5,6,15,16]. Furthermore, we have shown rescue of functional photoreceptor neurons using a recombinant AAV.PDE-b vector in the rd mouse model of retinal degeneration . In the present study, we have constructed rAAV vectors expressing (1) the murine NTN cDNA under the control of the CMV immediate early promoter/enhancer, named AAV.NTN (Figure 1A), and (2) the human GFRa-2 cDNA driven by 1.4 kb of the murine opsin promoter, named AAV.GFRa-2 (Figure 1B). In order to discriminate between transgene and endogenous gene expression, both recombinants incorporated epitope tags; respectively the paramyxovirus SV5 sequence for AAV.NTN at the C-terminal of the coding sequence, and the influenza HA sequence for AAV.GFRa-2, inserted after codon 32 of the GFRa-2 cDNA.
Expression of the two transgenes was initially evaluated in vitro in rd mouse retinal primary cell culture, which is a mixed population of cells expressing neuronal and glial markers [11,17]. Endogenous expression of NTN and GFRa-2 was detected in non-infected primary cell culture by immunohistochemistry (Figure 2A,B). For NTN, diffuse immunostaining throughout the retinal cell bodies (Figure 2A) suggested a cytoplasmic localization. In contrast, GFRa-2 immunolabelling was more concentrated at the cell periphery (Figure 2B) implying a membrane-bound distribution. No endogenous expression of V5 or HA epitopes was observed in the non-infected cells, and no fluorescence was detected in antibody-negative controls (data not shown). Additionally, none of the rAAV-infected cultures showed changes in cell morphology compared with controls.
In AAV.NTN-infected cells, co-localization of anti-NTN and anti-V5 immunoreactivity was observed in the cytoplasm and the neuronal processes, confirming transgene expression (Figure 2C,D). Similar to previous studies using AAV.CMV-lacZ , primarily neuronal but also glial cell types showed expression of the transgene by double immunolabelling using specific markers (data not shown). In AAV.GFRa-2 infected cells, GFRa-2 transgene expression was co-localized with HA immunostaining at the cell membrane level (Figure 2E,F), confirming transduction of retinal cells by this construct and demonstrating appropriate translocation of the receptor. In these cultures, transgene expression was almost exclusively detected in neuronal cells as determined by double immunolabelling using anti-neurofilament antibody (data not shown). This may reflect constraints on expression driven by the partial opsin promoter, which contains sequence elements sufficient for targeting expression specifically to photoreceptors in vivo [14,18-22].
Having confirmed the suitability of the two rAAVs for expressing exogenous NTN and GFRa-2 in retinal cells in vitro, we assessed the potential of the constructs for in vivo delivery following intraocular injections of rd mice according to previously described protocols . Co-localization of NTN and V5 immunolabelling confirmed NTN transgene expression in vivo in eyes injected with AAV.NTN (Figure 3A,C). Immunoreactivity was detected mainly at the outer segments of the surviving photoreceptors and to a lesser extent at the ganglion cell layer level. The distribution is in accord with the fact that the expression of NTN was under the control of the CMV promoter, which has previously been found to direct expression of reporter genes to these locations following intraocular injection .
GFRa-2 and HA immunostaining was detected only at the residual photoreceptor outer segments in the retinas of AAV.GFRa-2-injected mice (Figure 3E,G), demonstrating that the vector is capable of transducing the expression of GFRa-2 in vivo. These results also confirm that the 1.4 kb promoter segment of the photoreceptor-specific murine opsin gene efficiently directs the transgene expression to the photoreceptors. Previous studies have shown that both longer and shorter segments of the opsin promoter are effective elements for targeting the expression of various genes to mouse photoreceptors [14,18-22]. No NTN or GFRa-2 transgene expression was detected in the right eyes of injected animals (Figure 3D,H) or in animals injected with the virus resuspension buffer or with an AAV virus containing a reporter gene (data not shown). Endogenous expression of NTN was detected mainly at the ganglion cell layer, but not at the photoreceptor level in the non-injected eyes (Figure 3B). Low levels of endogenous GFRa-2 were diffusely detectable throughout the retina of the non-injected eyes (Figure 3F).
The potential effect of NTN and GFRa-2 gene expression on photoreceptor function was assessed by ERG testing (Figure 4). Non-injected 18 day-old control rd mice ERGs presented a barely-detectable a-wave (generated by the residual photoreceptors) and a late b-wave (generated by the inner retina) as described previously  (Figure 4A). Comparable ERG traces were observed for mice injected with the virus resuspension buffer PBSS or an AAV virus containing the lacZ reporter gene (at doses similar to AAV.NTN and AAV.GFRa-2), confirming that the injection process by itself did not have any non-specific effect on photoreceptor function, in agreement with our previous report  (Figure 4B,C). AAV.NTN and AAV.GFRa-2 injections did not significantly modify the ERG recordings, suggesting that the photoreceptor function was not improved (Figure 4D,E). Since we have previously shown a 17% photoreceptor function rescue after AAV.phosphodiesterase-b injection and assessment at similar time points , absence of rescue in this study is unlikely to be related to the promoter used or to the rapid degenerative process of the rd mouse model. High levels of neurotrophic factors may be needed to counteract the retinal degeneration, and injection of virus at higher doses would test this possibility. The present study indicates that expression of exogenous GFRa-2 or NTN in rd retinas (which show low NTN receptor levels; see also ), does not affect photoreceptor function, suggesting that simple modulation of the expression of these genes is insufficient to act therapeutically in this model. The complexity of neurotrophic interactions is highlighted by recent reports that combinations of growth factors from different families are required for neuronal survival [24,25]. The fact that only a partial protective effect was observed (1) in rd mice injected with adenoviral vectors containing ciliary neurotrophic factor (CNTF) , and (2) in rats injected with adenovirus vectors containing brain-derived neurotrophic factor (BDNF) after transection of the optic nerve , supports this hypothesis. In this context, combined injection of NTN with other neurotrophic factors (such CNTF or BDNF) may prove beneficial in reducing photoreceptor loss in multiple forms of retinal degeneration.
In conclusion, owing to the fact that these rAAV constructs were able to (1) transfer and express the two transgenes, (2) target expression through the use of different promoters, and (3) permit discrimination between endogenous and exogenous forms of the encoded proteins by the presence of dissimilar tags, they will constitute useful tools to assess the potential of NTN to rescue photoreceptors in synergy with other neurotrophic factors. Additionally, in view of the importance of NTN in the enteric and parasympathetic nervous systems [28,29], these or appropriately derived constructs could be examined in the context of therapy for autonomic neuropathies.
We gratefully acknowledge the support of this work by grants from the British Retinitis Pigmentosa Society, the Guide Dogs for the Blind Association, the Iris Fund for Prevention of Blindness, the National Lottery Charities Board, the University of London, and the Royal Society. The authors thank Ms. Hannah Stewart for technical support, Drs. Karen A. Vincent and Sam C. Wadsworth of Genzyme Corporation for their collaborative contributions, and Drs. Basil S. Pawlyk and Michael A. Sandberg of the Berman-Gund Laboratory for the Study of Retinal Degeneration for advice and suggestions on ERG procedures and methods. For the provision of clones, we thank Dr. N. Muzyczka of the University of Florida and Dr. J. Chen of the California Institute of Technology.
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