Brief Communication

Gene Therapy (2003) 10, 523–527. doi:10.1038/sj.gt.3301929

Intraocular gene delivery of ciliary neurotrophic factor results in significant loss of retinal function in normal mice and in the Prph2Rd2/Rd2 model of retinal degeneration

F C Schlichtenbrede1, A MacNeil1, J W B Bainbridge1, M Tschernutter1, A J Thrasher2, A J Smith1 and R R Ali1,2

  1. 1Department of Molecular Genetics, Institute of Ophthalmology, University College London, UK
  2. 2Molecular Immunology Unit, Institute of Child Health, University College London, UK

Correspondence: Dr RR Ali, Department of Molecular Genetics, Institute of Ophthalmology, University College, Bath Street, London ECIV 9EL, UK

Received 1 July 2002; Accepted 4 October 2002.

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Abstract

Intraocular delivery of a variety of neurotrophic factors has been widely investigated as a potential treatment for retinal dystrophy (RD). The most commonly studied factor, ciliary neurotrophic factor (CNTF), has been shown to preserve retinal morphology and to promote cell survival in a variety of models of RD. In order to evaluate CNTF as a potential treatment for RD, we used the Prph2Rd2/Rd2 mouse. CNTF was expressed intraocularly using AAV-mediated gene delivery either by itself or, in a second treatment group, combined with AAV-mediated gene replacement therapy of peripherin2, which we have previously shown to improve photoreceptor structure and function. We confirmed in both groups of animals that CNTF reduces the loss of photoreceptor cells. Visual function, however, as assessed over a time course by electroretinography (ERG), was significantly reduced compared with untreated controls. Furthermore, CNTF gene expression negated the effects on function of gene replacement therapy. In order to test whether this deleterious effect is only seen when degenerating retina is treated, we recorded ERGs from wild-type mice following intraocular injection of AAV expressing CNTF. Here a marked deleterious effect was noted, in which the b-wave amplitude was reduced by at least 50%. Our results demonstrate that intraocular CNTF gene delivery may have a deleterious effect on the retina and caution against its application in clinical trials.

Keywords:

CNTF, neuroprotection, rds mouse, retinal degeneration, viral vector, gene therapy

Photoreceptor dystrophies are one of the commonest causes of inherited blindness in the Western world. They may result from a defect in any one of over 60 different genes, many of which are photoreceptor specific.1 A common feature of these diseases is the loss of photoreceptors by apoptosis during the degeneration. Since, to date, no treatment is available for these conditions much effort has been directed towards the development of new treatment strategies, including gene therapy. A number of groups have recently reported successful gene replacement therapy using viral vectors in animal models of recessive disease.2,3 An alternative strategy, the delivery of genes encoding neurotrophic factors (here referred to as neuroprotection) has been investigated as a potential treatment, particularly for dominant disease. Treatments involving one of a number of neurotrophic factors, such as ciliary-derived neurotrophic factor (CNTF), fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), have been shown to promote photoreceptor cell survival in a variety of retinal degeneration models.4,5,6,7,8,9,10 The cytokine CNTF has been the most widely evaluated of these factors. Initial trials involving intravitreal injections of the recombinant protein into rodent models of recessive disease demonstrated slowing of photoreceptor loss in the rd and Prph2Rd2/Rd2 mouse.5 The short half-life of the recombinant protein in vivo prompted repeat injections in larger animal models,8 the development of ocular inserts (pharmacological slow release devices) and finally gene therapy approaches. Cayouette and Gravel11 reported photoreceptor loss in the retinal dystrophy (RD) mouse was delayed by 18 days following intravitreal injection of an adenoviral vector expressing CNTF. Intravitreal injections of the same vector in 3-week-old Prph2Rd2/Rd2 mice resulted in photoreceptor loss being delayed for up to 7.5 weeks after treatment. In this study, they also reported a very slight improvement in a- and b-wave ERG amplitude.12 Following intravitreal injection of an AAV vector carrying a gene encoding CNTF, Liang et al9 showed preservation of photoreceptors at 9 months after treatment in the Prph2Rd2/Rd2 mouse and two rhodopsin transgenic rat models of dominant disease at 8 months after treatment. ERGs at this late time point suggested there was little difference in function between treated animals and untreated controls. In similarly treated rhodopsin transgenic rats, the ERGs were reduced at 6 months after injection. The same group has also used the vector to slow photoreceptor cell loss in mice that were homozygous for a targeted disruption of the rhodopsin gene.10 Thus, a number of studies have demonstrated the effect of vector-mediated CNTF gene expression on photoreceptor cell loss, suggesting that this may be an effective treatment for human retinal dystrophies. These studies have focused on animals in which there is very little function and concentrated on the morphology of the retina. A recent report, however, has evaluated long-term AAV-mediated CNTF gene expression in a transgenic mouse model of retinal disease caused by a dominant mutation in the Prph2 gene, in which retinal function is relatively normal and only declines slowly with photoreceptor cell loss. Bok et al13 found a 23% preservation of photoreceptor cells as compared to the untreated side at 5 months after treatment, but observed reduced ERG recordings following treatment.

In this study, we wanted to determine the effect of CNTF gene expression in the Prph2Rd2/Rd2 mouse in combination with gene replacement therapy. The Prph2Rd2/Rd2 mouse has proven to be a very useful tool for assessing the efficacy of gene therapy protocols for retinal dystrophies. Owing to the failure to develop photoreceptor outer segments, the Prph2Rd2/Rd2 mouse has extremely limited ERG responses in untreated adult animals, allowing easy quantification of functional improvements following treatment. We have previously demonstrated improvement of photoreceptor ultrastructure and function in the Prph2Rd2/Rd2 mouse following AAV-mediated expression of Prph2.2 A detailed evaluation of the effects of treatment on cell survival revealed, however, that the rate of photoreceptor cell loss is not slowed.14 In this study, we wanted to determine whether we could improve photoreceptor survival as well as improving function by combining neuropro-tection with gene replacement. We therefore injected AAV carrying a CNTF gene driven by a CMV promoter (AAV.CMV.CNTF) subretinally together with AAV carrying a Prph2 gene driven by a rhodopsin promoter (AAV.rho.rds) in postnatal day (P10) in Prph2Rd2/Rd2 mice. As controls, we injected each vector separately in Prph2Rd2/Rd2 mice. We also injected adult wild-type mice with AAV.CMV.CNTF in order to test the effects of CNTF on fully developed nondegenerating retina.

A murine CNTF cDNA fused to a fragment of the first 20 amino acids of mouse prepro-nerve growth factor as secretion signal (kind gift from S Fauser, University Tuebingen, Germany) was cloned between the CMV promoter and the GFP gene in the construct AAV.CMV.GFP, described by Zhang et al,15 together with an internal ribosomal entry sequence (IRES) to form pD10/CMV-CNTF-IRES-GFP. The cDNA was sequenced to ensure that no mutations had been inadvertently introduced. The second construct, encoding Prph2 driven by a rhodopsin promoter (pD10/rho-peripherin2), has been previously described.2 The constructs were used for the production of AAV.CMV.CNTF and AAV.rho.rds according to a previously described protocol.15 We performed subretinal injections in adult CBA mice and in Prph2Rd2/Rd2 mice on p10 as previously described.2 Briefly, the needle tip of a microsyringe was placed in the subretinal space of the right eye under direct ophthalmoscopic control and approximately 1 mul of viral suspension was injected to produce a bullous retinal detachment covering about 30% of the fundus. Two separate injections in opposing quadrants were performed to increase the treatment area to around 60%. To confirm CNTF expression after AAV infection, a specific ELISA assay was used to demonstrate highly increased CNTF levels in tissue culture supernatants from 293T cells 3 days after AAV infection (5.5 mug/ml CNTF in supernatant from transduced cells versus 0.04 mug/ml in controls). In pooled whole eye homogenates taken 20 weeks after subretinal injection of AAV.CMV.CNTF, we were able to detect a 2.7-fold increase in the level of CNTF compared with the levels in uninjected contralateral eyes (4.58 versus 1.7 mug/ml). Sample eyes from CBA mice were cryosectioned 12 weeks after injection and examined for GFP expression, which was located, as expected, predominantly in the photoreceptor cell layer and RPE (data not shown).16

ERGs from treated Prph2Rd2/Rd2 and wild-type mice were recorded in a standardised fashion at various time points up to 19 weeks after treatment by the same investigator (FCS). All recordings were obtained simultaneously from both eyes of the animal in order to provide an internal control. For statistical analysis all values were paired (R treated and L untreated), thus controlling for the interanimal and test–retest variability in rodent ERGs. The b-wave amplitude in the ERG of untreated Prph2Rd2/Rd2 mice is very low compared with a wild-type animal (typically around 70 muV at 3 weeks versus around 400 muV); this diminishes fairly rapidly to about 50% in the first 2 months, which is reflected histologically in a loss of 50% photoreceptors during the same period. Changes in b-wave amplitude were assumed to be significant at p-values <0.05. In ERG recordings of Prph2Rd2/Rd2 mice (n=16) treated with AAV.CMV.CNTF, a marked reduction of the b-wave amplitude on the treated side was noted. This effect was significant at two time points, 6 and 9 weeks, (Figures 1a and 2a). A control group of Prph2Rd2/Rd2 mice (n=10) treated with AAV.rho.rds alone showed markedly improved traces on the treated side, with significantly higher b-wave amplitudes at 4 time points (Figures 1b and 2b). When Prph2Rd2/Rd2 mice (n=12) were treated with a combination of AAV.rho.rds and AAV.CMV.CNTF, no difference between treated and untreated animals were found at any time point (Figures 1c and 2c). Thus, additional treatment with vector expressing CNTF appeared to negate the improvement in function normally seen following gene replacement therapy alone. Subretinal injection of AAV.CMV.CNTF in wild-type CBA mice (n=7) resulted in significantly reduced b-wave amplitudes as compared with untreated wild-type animals at all time points recorded (Figures 1d and 2d). This was approximately 50% of the normal amplitude at 8 weeks after treatment and declined still further thereafter.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

ERG recordings following gene delivery. A representative intensity series of right and left eyes is shown at single time points after treatment. (a) 6 weeks following subretinal injection of Prph2Rd2/Rd2 mice with AAV.CMV.CNTF alone, there is a marked reduction in the b-wave amplitude compared to the untreated side. (b) Gene replacement therapy with AAV.rho.rds improves function, with a marked increase in b-wave amplitude 6 weeks after treatment and with the pattern of the trace resembling those from normal animals. (c) Subretinal injection of a combination of AAV.rho.rds and AAV.CMV.CNTF does not lead to an improvement in function. Traces shown were recorded 9 weeks after treatment. (d) Subretinal injection of AAV.CMV.CNTF in the wild-type (CBA) mouse leads to diminished ERG traces with markedly reduced amplitudes. Traces shown were recorded 12 weeks after treatment. All animals were dark-adapted overnight (16 h) and Ganzfeld ERGs were obtained simultaneously from both eyes of each animal. In all animals, the right eye (R) was injected with the left eye (L) serving as an uninjected internal control. All procedures for recording were carried out under dim red light. The mice were anaesthetised using a single intraperitoneal injection of ketamine (70 mg/kg), xylazine (12 mg/kg) and atropine (1 mg/kg). The pupils were dilated using one drop each of Phenylephrine 2.5% and Tropicamide 1% to each eye. ERGs were recorded using commercially available equipment (Toennies Multiliner Vision, Jaeger/Toennies) after placing surface corneal ring electrodes and subdermal reference and ground electrodes. Bandpass filter cutoff frequencies were 1 and 300 Hz for all measurements. Single flash recordings were obtained at light intensities increasing from 0.1 to 3000 mcds/m2. In all, 10 responses per intensity level were averaged with an interstimulus interval of 5 s (0.1, 1, 10 and 100 mcds/m2) or five responses per intensity with a 17 s interval (1000 and 3000 mcds/m2). The data were analysed and stored using the Toennies Multilinear Vision program. Animals were analysed at weekly intervals over a period of several months. Here, representative recordings are presented from time points at which the difference between treated (R) and untreated (L) eyes is greatest (see Figure 2), with increasing stimulus intensity from top to bottom in each panel.

Full figure and legend (59K)

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Averaged ERG b-wave amplitudes at various time points following gene delivery. (a) Following subretinal injection of Prph2Rd2/Rd2 mice with AAV.CMV.CNTF alone, there is a significant reduction in the b-wave amplitude at 6 and 9 weeks after injection. (b) Gene replacement therapy with AAV.rho.rds improves function, with a significant increase of b-wave amplitude at four time points up to 8 weeks postinjection. (c) Subretinal injection of a combination of AAV.rho.rds and AAV.CMV.CNTF does not lead to an improvement in function compared to the untreated side since b-wave amplitudes are never significantly different between R and L eye. (d) Subretinal injection of AAV.CMV.CNTF in the wild-type (CBA) mouse leads to diminished ERG traces with significantly reduced amplitudes at three different time points. For each treatment group, an ERG intensity series was recorded, as described in legend to Figure 1, over the time course indicated in the panels. For each animal, the b-wave amplitude at 100 mcds/m2 was used for statistical analysis. The b-wave values (a-wave trough to b-wave peak) of the treated (R) eye were paired with the untreated (L) contralateral eye to provide an internal control. A paired Student's t-test was used to evaluate significance (p<0.05). All calculations were carried out using standard spreadsheet software. This method controls for interanimal- and test–retest variance apparent in rodent ERGs. The error bars indicate the SD and stars denote a significant difference in a paired Student's t-test between R and L eye.

Full figure and legend (180K)

Seven weeks after injection of Prph2Rd2/Rd2 mice and 18 weeks after injection of wild-type animals, three to four animals were killed per treatment group and the eyes were examined morphologically following semithin sectioning (Figure 3). To determine preservation of outer nuclear layer (ONL) thickness, two areas at 250 mum distance from the optic nerve head were identified on the corresponding slides, and rows of nuclei in the ONL were counted on four sections per eye by two independent observers. The effect of AAV.rho.rds treatment on Prph2Rd2/Rd2 mice is illustrated in Figure 3a and c. Prph2Rd2/Rd2 mice treated with AAV.CMV.CNTF alone or in combination with AAV.rho.rds showed on an average 5.5 or 5.25 rows of nuclei in the ONL, respectively (Figure 3d and b) as compared to 3.5 rows in untreated Prph2Rd2/Rd2 mice (Figure 3c) or Prph2Rd2/Rd2 mice treated with AAV.rho.rds alone (Figure 3a). For all CNTF treatment groups, the difference in rows of nuclei in the ONL is significantly greater, with all p-values <0.02. Retinal tissue organisation appeared to be reduced (ie the ONL appears to be less densely packed and rows of nuclei are less regular) in all Prph2Rd2/Rd2 animals injected with AAV.CMV.CNTF (Figure 3b and d) compared with either Prph2Rd2/Rd2 mice injected with AAV.rho.rds (Figure 3a) or uninjected animals (Figure 3c). We could, however, still observe to some extent outer segment material in Prph2Rd2/Rd2 mice injected with a combination of AAV.CMV.CNTF and AAV.rho.rds (Figure 3b). Wild-type mice have around nine rows of densely packed nuclei in the ONL (Figure 3e), but following treatment with CNTF, even though there was no photoreceptor cell loss (P=0.5), the ONL is less densely packed with less regular rows (Figure 3f).

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Histological analysis of retinae following gene delivery. Prph2Rd2/Rd2 mice were killed 7 weeks after subretinal injection. (a) In Prph2Rd2/Rd2 mice injected with AAV.rho.rds, newly formed outer segments are observed, but the ONL thickness is unchanged. (b) In Prph2Rd2/Rd2 mice injected with a combination of AAV.CMV.CNTF and AAV.rho.rds, there is some outer segment material as well as ONL preservation. (c) In untreated Prph2Rd2/Rd2 mice, the ONL is thinner and there is a lack of outer segments. (d) In Prph2Rd2/Rd2 mice injected with AAV.CMV.CNTF, the ONL is preserved (the detachment is a processing artefact). Wild-type mice were killed 18 weeks after injection. (e) Normal retina from wild-type mouse. (f) Following subretinal injection of AAV.CMV.CNTF in wild-type mice, the ONL is less well organised and the outer segments appear to be shorter. All eyes were surgically removed and resin embedded for semithin sectioning at 0.7 mum according to a standard protocol.14 Sections were stained with toluidin blue and evaluated using a Leitz Diaplan microscope fitted with a Leica digital camera DC 500 for image capture. Retinal layers: ONL=outer nuclear layer, IS=inner segments, and OS=outer segments.

Full figure and legend (217K)

Our results confirm that CNTF gene delivery enhances photoreceptor cell survival in the Prph2Rd2/Rd2 degeneration model. However, in contrast to the study by Cayouette et al,12 we could find no improvements in retinal function as assessed by ERG following CNTF gene delivery. Rather than improving function when neuroprotection was used, either as a single strategy or in combination with the gene replacement therapy, func-tion was significantly decreased. Furthermore, wild-type mice showed a marked reduction in b-wave amplitudes following treatment with AAV.CMV.CNTF. The alteration in ERG trace may reflect the morphological changes caused by CNTF gene expression. The changes we have observed – a less well-ordered ONL and, in animals also treated with a gene replacement vector, less outer segment material – are consistent with recent reports that suggest that exposure to high levels of CNTF may have an impact on photoreceptor differentiation.13,16 However, CNTF has been shown to exert an indirect effect on photoreceptor cell survival through cells of the inner retina and Mueller cells. Wahlin et al have demonstrated that following intra-ocular injection of recombinant CNTF, signalling pathways are activated in Mueller, ganglion and amacrine cells, but not photoreceptors.17 This effect was found both in normal and degenerating rodent retinae.18 It is the inner retinal neurons and Mueller cells that are responsible for generating the b-wave. Thus, the effect of CNTF on these cells may also be counteracting the potential benefit of photoreceptor preservation. Furthermore, CNTF overexpression might stimulate remodelling of the inner retina, leading to a change in cell function, a decrease in b-wave amplitude and subsequently to dedifferentiation of the photoreceptor cell, as reflected by the changes in chromatin staining.13 It has been suggested that in the course of retinal degeneration, loss of input from photoreceptors leads to input-dependent secondary neurons of the inner retina 'seeking replacement' for the input of lost photoreceptors.19 It would appear that the remodeling of the inner retina that normally occurs in the course of degeneration is exacerbated by the expression of CNTF.

Our study shows the intraocular treatment with CNTF may result in unwanted side effects. These effects might be tolerable provided CNTF administration is only temporary. This might be achieved through the use of an inducible promoter or a pharmacological slow release device with later removal of the ocular insert. To achieve a sustained morphological rescue without causing retinal damage a fine balance would have to be reached, requiring detailed dose–response studies, further complicated by the relatively high individual variability in the degenerating retina. Alternatively, other neurotrophic molecules should be evaluated for safety and efficacy. In retinal dystrophies, the degenerating retina undergoes a highly complex remodelling process, where any treatment-induced negative stimuli must be avoided.

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Acknowledgements

FCS is a Marie Curie Fellow (EU). This work was supported by grants from The Sir Jules Thorne Charitable Trust and The Foundation Fighting Blindness.