Gene transfer into the mouse retina mediated by an adeno-associated viral vector
Gene transfer into the mouse retina mediated by an adeno-associated viral vectorRobin R. Ali*, Martin B. Reichel, Adrian J. Thrasher1, Roland J. Levinsky1, Christine Kinnon1, Naheed Kanuga, David M. Hunt and Shomi S. Bhattacharya
Department of Molecular Genetics, Institute of Ophthalmology, University College London, Bath St., London EC1V 9EL, UK and 1Division of Cell and Molecular Biology, Institute of Child Health, University College London, Guilford St., London WC1N 1EH, UK
Received January 26, 1996;Revised and Accepted February 26, 1996
Gene transfer to photoreceptor cells may provide a means for arresting the retinal degeneration that is characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). However, transduction of photoreceptors has to date been inefficient, and further limited by toxicity and immune responses directed against vector-specific proteins. An alternative vector system based on adeno-associated virus (AAV) may obviate these problems, and may be useful for transduction of neuronal cells. In this study we have demonstrated successful transduction of all layers of the neuroretina as well as the retinal pigment epithelium (RPE) following subretinal injection of recombinant AAV particles encoding lac Z. Furthermore, the efficiency of transduction of photoreceptors is significantly higher than that achieved with an equivalent adenoviral vector. This is the first report showing that AAV is capable of transducing photoreceptor cells and supports the use of this vector system for gene therapy of retinal diseases such as RP.
Inherited forms of retinal degeneration are relatively common causes of blindness in adult life (1 ). Of these, retinitis pigmentosa (RP) is the best characterised. Five different non-syndromic RP genes have been identified to date (2 -6 ) all of which are expressed exclusively in photoreceptor cells. However, photo- receptor cells and the retinal pigment epithelium (RPE) are in close proximity and are inter-dependent (the anatomy of the retina is shown in Fig. 1 a). Future research may reveal genes that are expressed in the RPE and which play a role in some forms of retinal degeneration [the Royal College of Surgeons' rat, for instance, has a single gene defect of the RPE which results in retinal degeneration (7 )]. Delivery of a normal gene or gene product to these cells may arrest the degenerative process and thus preserve vision.
In order to determine the efficiency of transduction of AAV in comparison to AV, and to exclude complications arising from the immunogenicity of the adenoviral backbone, or from the bacterial reporter gene lac Z, studies were conducted in immunodeficient nude mice. An adeno-associated viral vector with an internal CMV promoter-driven lac Z gene (AAV.CMVlacZ) at a titre of 107 infective units (iu) per ml was injected subretinally (Fig. 1 b) into adult nude mice resulting in detachments encompassing approximately 25% of each retina. The animals were sacrificed 28 days after the procedure. X-gal staining was observed only in the area corresponding to the retinal detachment around the site of injection. Within this area between 100 and 500 transduced photoreceptors were found either in small clusters or scattered as single cells (Fig. 2 a) in 50% of the eyes examined (n = 10). A total of 1100 positive photoreceptor cells were counted. In some cases we see staining throughout the photoreceptor: in the nucleus, cell body, inner and outer segments (Fig. 2 b). Observation of positive photoreceptors without staining of associated rod outer segments (ROS) may be a consequence of lower levels of [beta]-gal production in these particular cells, or a consequence of the angle of sectioning. Four weeks after injection of 2 [mu]l of AAV at a titre of 1 * 107 iu/ml, there was no evidence of direct toxicity. The proportion of transduced photoreceptor cells is significantly higher than with adenovirus containing a similar CMV promoter-driven lac Z reporter gene (AV.CMVlacZ). Only one positive photoreceptor cell was observed in all the eyes examined (n = 22) following subretinal injection of AV.CMVlacZ at a titre of 5 * 108 plaque forming units (pfu) per ml.
After injection with AAV.CMVlacZ, positive cells were also seen in other layers of the neuroretina: the ganglion cell layer (3rd neuron) as well as the bipolar cell layer, which includes the bipolar cells (2nd neuron) as well as Müller, amacrine and horizontal cells (Fig. 2 c). In addition to the transduced neuroretina, there were many positive RPE cells-up to 25% of the total-along with positive staining in the adjacent ROS layer (Fig. 2 d). A similar pattern of staining in the RPE and ROS layer is also observed after subretinal injection with AV.CMVlacZ (Fig. 2 e). The staining observed in the ROS layer may result from detached RPE microvilli or be due to secretion or diffusion of [beta]-gal from transduced RPE. This is supported by an additional experiment using an adenoviral vector containing a lac Z gene with a nuclear localisation signal. Following subretinal injection of AV.CMVlacZnuc (109 pfu/ml) we were unable to see any positive photoreceptor cell nuclei, even though the ROS layer was still stained (Fig. 2 f). However, positive staining was seen in the RPE cell nuclei. This indicates that only the RPE cells have been transduced.
In this study, we have demonstrated that AAV vectors mediate efficient transduction of retinal cells. Furthermore, transduction of photoreceptors is over two thousand-fold more efficient than that achieved with conventional adenovirus-based systems and is not associated with direct toxicity after one month. Therefore, adeno-associated viral vectors, although requiring further development, offer a promising way of targeting photoreceptor cells. Improvements in the production of AAV (allowing higher titres) along with alternative delivery methods that increase local concentrations are likely to raise substantially the proportion of transduced photoreceptor cells. However, it is not yet clear how many photoreceptors will have to be transduced in order to provide a significant restoration of function. For example, rescue of scattered photoreceptor cells may increase light perception, whilst in eyes with a fovea, focal targeting may enhance central vision. Transduction of the non-neuronal retinal pigment epithelium is particularly efficient and similar to that experienced using AV vectors. The intrinsic phagocytic activity of this layer may mediate efficient uptake of viral particles. Delivery of therapeutic genes to these cells may become useful for both inherited and acquired retinal pathology.
AAV.CMVlacZ (pTRCMV[beta],N.M.) was packaged into infectious particles by co-transfection of a 293 cell line and infection with helper wild-type adenovirus type 5, as previously described (13 ). The AAV vector was purified and separated from adenovirus by sequential caesium chloride banding and finally concentrated by ultrafiltration in a Microcon® concentrator (Amicon Ltd., Stonehouse, Gloucestershire). Infectious titre of AAV preparations was determined by transduction of HeLa cells. AV stocks were prepared from infected 239 cell lysates, purified by sequential caesium chloride banding and titred by conventional plaque assay.
Mice were anaesthetised by ip injection of 0.2 ml Hypnorm (Janssen Pharmaceutical Ltd, Oxford), and Hypnovel (Roche, Welwyn Garden City) mixed 1:1:6 with distilled water. Following dilation with 1% tropicamide [1% Mydriacyl, Alcon Labs (UK) Ltd., Hemel Hempstead], the eyes were gently protruded using a rubber sleeve and subsequently covered with 2% hydroxypropylmethylcellulose in PBS and a cover slip. This allowed surgery to be performed under direct retinoscopy through an operating microscope. The tip of a 1.5 cm, 34-gauge hypodermic needle [Hamilton (GB) Ltd., Dundee] was brought into focus between the retina and RPE and approximately 2 [mu]l of PBS or viral suspension containing AAV.CMVlacZ, AV.CMVlacZ or AV.CMVlacZnuc was injected to produce a retinal detachment. All the mice [BALB/cOlaHsd-nu/nu (Harlan Olac Ltd., Bicester)] were injected on the same day by the same experienced operator and the procedure assessed for consistency by direct retinoscopy. The animals were 8 weeks old at the time of injection (+- 1 week).
Animals were killed by cervical dislocation and the enucleated eyes were prefixed by immersion in 10% buffered formalin for 45 min. The cornea and lens were removed and the eye cups rinsed in PBS and incubated overnight at room temperature with X-gal (5-bromo-4-chloro-3-indolyl-[beta]-D-galactopyranoside; Calbiochem, La Jolla Ca) in a solution containing 10 mM K3Fe(CN)6, 10 mM K4Fe(CN)6, 2 mM MgCl2, in PBS.
After incubation with X-gal, the tissue was fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned at a thickness of 5-10 [mu]m. Sections were counterstained with nuclear fast red and examined by light microscopy.
We thank Nicholas Muzyczka for the AAV.CMV lacZ plasmid. This work was supported by the Medical Research Council. M.B.R is supported by Deutsche Forschungsgemeinschaft grant Re1121/1-1 and A.J.T. by the Wellcome Trust.
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