Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

sity during light cycle = 700 ± 50 lux, Sekonic L-28C photometer, NY, USA). After the 6 days, the light cycle was reversed, (12 : 12 h dark-light). The 180° change in photoperiod (light reversal) was made such that the mice were initially exposed to 24 h of dark, as a result of 2 consecutive 12 h dark periods. Data was recorded until the circadian rhythms of the mice entrained to the new lighting conditions.

2.3. Data Analysis

Data bins were collected from each animal and graphed as double plot actograms for visual inspection. Hourly activity data was analyzed in blocks of 72 hourly bins (3 days). The spectral amplitudes (in natural log units) of the possible periods (and their Z score transformations) of each block (72 h) were determined for each mouse independently using the periodogram method of Spectral analysis (SPSS-PC). Z scores for the 24 h period were analyzed further to determine if significant 24 hour rhythms were present. Individual activity amplitudes and acrophases were generated using Halberg’s cosinor regressions (with period = 24 h).14

Parametric statistical analysis of the amplitudes, acrophases and 24 h z-scores across days were performed with the general linear model (GLM) Analysis of Variance (ANOVA) procedure for repeated measures, which including simple pair-wise contrast tests for further group comparisons, using SPSS 10.0 for Windows, with a = 0.05. For a more detailed discussion of the statistical analysis see Daniels et al. 20034 and associated papers.15,16

3. RESULTS

Periodogram analysis showed all groups of mice maintained a significant 24 h period in their locomotor circadian activity (data no shown). Double plot actograms of the data collected showed that, while all 3 groups of mice were capable of re-entraining after the 12 : 12 h light reversal,17,18 the amount of time taken to re-entrain varied between the groups. The re-entrainment of C57 controls to the photoperiod reversal was rapid, being complete by 4 days in all mice (Fig. 34.1A), while re-entrainment of the control Rpe65-/- group took significantly longer, needing at least 13 days (Fig. 34.1B). While there was little withinsubjects variance in the two control groups, the re-entrainment times of the injected Rpe65-/- group varied considerably; mice in this group taking between 8 & 14 days to reentrain (Fig. 34.1C, 1D).

When the amplitude data for each group was compared using simple contrast tests, the control Rpe65-/- mice expressed a hypolocomotive phenotype with respect to the C57 controls (p = 0.014). Assessment of the injected Rpe65-/- group showed some of the animals (n = 3) exhibited a small amount of recovery from the Rpe65-/- hypolocomotive phenotype, however a majority of the group displayed the same phenotype as the control Rpe65-/-s (data not shown, C57 p = 0.213; control Rpe65-/- p = 0.182).

The acrophase of a circadian rhythm shows the peak of the 24 hour cycling component of activity, occurring in the dark phase of the photoperiod for motor activity in nocturnal animals. By tracking the change in acrophase following light reversal, the re-entrainment rate of the circadian rhythm can be assessed. Upon analysis, the C57 control group exhibited rapid re-entrainment, which differed significantly from both the control and injected

improvements, both with GT treatments and their in vivo assessments, will work towards clinical benefits for RD patients.

5. ACKNOWLEDGEMENTS

The authors would like to thank Dr Mela Brankov, Dr Wei-Yong Shen and Mr Stephen Moore, Lions Eye Institute, Perth, Western Australia for their technical expertise. The Rpe65-/- knockout mice11 and goat anti mouse RPE65 antibody13 were kindly provided by Dr T. Michael Redmond, National Eye Institute, National Institute of Health, Bethesda, MD, USA. This project was funded in part by the National Health and Medical Research Council of Australia and Retina Australia.

6.REFERENCES

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P.E. Rakoczy, Functional and structural recovery of the retina after gene therapy in the RPE65 null mutation dog, Invest. Ophthalmol. Vis. Sci. 44(4), 1663-1672 (2003).

7.K. Narfstrom, M. L. Katz, M. Ford, T. M. Redmond, E. Rakoczy and R. Bragadottir, In vivo gene therapy in young and adult RPE65-/- dogs produces long-term visual improvement, J. Hered. 94(1), 31-37 (2003).

8.R. J. Lucas, M. S. Freedman, M. Munoz, J. M. Garcia-Fernandez and R. G. Foster, Regulation of the mammalian pineal by non-rod, non-cone, ocular photoreceptors, Science 284(5413), 505-507 (1999).

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K.Pfeifer, RPE65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle, Nat. Genet. 20(4), 344-351 (1998).

12.P. E. Rakoczy, C. M. Lai, M. J. T. Yu, D. M. Daniels, M. Brankov, B. C. Rae, C. W. Stoddart, N. L. Barnett,

M.T. Martin-Iverson, T. M. Redmond, K. Narfstrom, X. Zhou and I. J. Constable, in: Retinal degeneration mechanisms and experimental therapy, edited by J. G. Hollyfield, M. M. La Vail (Luwer Academic / Plenum Publishers, New York, 2003), pp. 431-438.

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of feasibility of this approach has indeed already been established in dogs bearing a spontaneous mutation in the RPE65 gene (Acland et al. 2001; Narfström et al. 2003), as well as in knock-out mice (Dejneka et al. 2004; Lai et al. 2004). These studies have shown that an adeno-associated virus (AAV)-derived vector is able to deliver the RPE65 gene to RPE cells and thus to restore vision at least partially. Nevertheless, before a clinical trial can take place, a great effort must be provided to assess the bio-safety of the procedure. In particular, transgene expression has to be tightly controlled to achieve the following criteria: (i) expression should occur only in RPE cells; (ii) expression should reach the therapeutic level without disturbing the homeostasis of the target cells.

Adenovirus, AAV and lentivirus-derived vectors have been successfully used to transfer genes into retinal cells in vivo. Although adenoviruses are able to transduce RPE cells with a high efficiency, and photoreceptors to a lesser extent (Bennett et al. 1994; Li et al. 1994), they trigger an immune response which leads to the rejection of the transduced cells (Hoffman et al. 1997; Kumar-Singh and Farber 1998; Reichel et al. 1998). AAV-derived vectors are able to transduce RPE cells and photoreceptors, or photoreceptors alone, depending on the serotype, and allow for long-lasting transgene expression (Ali et al. 1996; Bennett et al. 1997; Flannery et al. 1997). Nevertheless, the delay that has been occasionally observed between AAV administration and transgene expression (Bennett et al. 1999) could become a serious hurdle in the case of LCA where treatment efficiency is desirable as early as in neonate. Furthermore, in the case of RPE65 mutations, the treatment by gene transfer will certainly have to remain active for the entire lifespan, a prerequisite that will be more likely fulfilled with an integrative vector. The lentivirus-derived vector is such a candidate, because its integrating properties make it particularly interesting for a “life-long treatment”. This type of vector is well known to target RPE cells in a highly predominant fashion after administration by injection into the subretinal space (Miyoshi et al. 1997; Kostic et al. 2003).

To develop a vector specifically designed for the gene therapy of RPE65 mutations in human, we have constructed a lentivirus in which the expression of the transgene is driven by a 0.8 kb proximal fragment of the human RPE65 promoter (LV-R0.8). We report here the transgene expression pattern of this vector in the mouse eye as well as the ability of this vector to trigger therapeutic levels of RPE65 protein in the RPE65 knock-out mouse model.

2. METHODS

2.1. Construction of Lentiviral Vectors

The lentiviral backbones used in this study were derived from the Hlox-EFS-GFP vector described in (Salmon et al. 2000). In the control vector (LV-R0.8-GFP), the EFS promoter was replaced by a fragment of 0.8 kb of the human RPE65 promoter. In the therapeutic vector (LV-R0.8-RPE65), after addition of the R0.8 promoter, the GFP coding sequence was replaced by the mouse RPE65 cDNA. Viral particles were produced by transient transfection of 293T cells as previously described (Naldini et al. 1996). Briefly, 293T cells were cotransfected by the vector plasmid, an HIV-1 packaging plasmid and an envelope plasmid encoding VSV-G. Two days after transfection, recombinant viral particles were harvested in the supernatant and concentrated by ultracentrifugation. Viral stocks were then stored in small aliquots at -80°C until use. The concentration of total viral particles was determined using ELISA quantification of the p24 capsid protein. Infectious activity was assessed on

infected 293T cells by flow cytometry detection of GFP-positive cells for the LV-R0.8-GFP vector and by western blot detection of RPE65 for the LV-R0.8-RPE65 vector.

2.2. Animal Treatment

All the mice used in this study were from the RPE65 knock-out line (Redmond et al. 1998). For vector injection, animals were sedated by volatile anesthesia; the temporal part of the sclera was carefully freed from the conjunctive tissue and perforated with a thin needle. A 31G Hamilton syringe was then inserted in the subretinal space and 1 ml of virus was injected.

For measurement of the corneal electroretinogram (ERG), mice were dark-adapted overnight and, under dim red light, anesthetized using ketamine and xylazine and placed in a Ganzfeld bowl. Pupils were dilated and thin silver wires were used as electrodes. Mice were then subjected to light stimuli as described in Grüter et al. (Gene Ther, in press).

2.3. Histology

To evidence transgene expression, animals were sacrificed by injection of pentobarbital, the eyes were then enucleated and fixed by immersion in PBS containing 4% paraformaldehyde. Eyes were then cut at 14 mm thickness on a cryostat and sections were collected on slides. Control transgene expression was evidenced by visualization of direct GFP fluorescence or GFP immunolabeling. RPE65 transgene expression was detected using a polyclonal rabbit antibody. Immunolabeling was performed as previously described (Kostic et al. 2003).

3. RESULTS AND DISCUSSION

3.1. In Vitro Testing of Lentiviral Vectors

We first assessed that our lentiviral vectors were able to transduce cells in vitro. To that aim, we used 293T cells, in which the R0.8 promoter in the lentiviral context is active. Three days after infection by LV-R0.8-GFP, cells were harvested and analyzed by flow cytometry. This allowed us to evidence a population of GFP expressing cells (data not shown), reflecting a titer of 5.10exp8 transducing units per ml of viral solution (TU/ml).

The activity of the LV-R0.8-RPE65 vector was assessed by Western blot analysis. We detected a band corresponding to RPE65 in cytosolic extracts of infected 293T cells, as well as in those of 293T cells transfected by the vector plasmid, the higher intensity of the band in the latter probably reflecting the higher copy number of transgenes per cell with the transfection method (Fig. 35.1).

3.2. Activity of the R0.8 Promoter in the Mouse Retina

We then investigated the pattern of transgene expression of the LV-R0.8-GFP vector in the eye of adult RPE65 knock-out mice. After subretinal injection into the adult, GFP was exclusively detected in the RPE cells across a wide span of the retina (Fig. 35.2a),

Figure 35.1. Western blot analysis of RPE65 expression. 293T cells were infected with LV-R0.8-RPE65 and cytosolic extracts were prepared three days later. For each condition, 25 mg proteins were loaded on SDS-PAGE and subsequently transferred to a PVDF membrane. RPE65 was detected using a rabbit polyclonal antibody, a HRP-linked secondary antibody (Amersham), and the ECL+ detection kit (Amersham). 1: uninfected cells; 2: cells transfected with the vector plasmid; 3: cells infected with the LV-R0.8-RPE65 vector.

Figure 35.2. Immunolabeling of GFP and RPE65 one week after injection of LV-R0.8-GFP (A-E) or LV-R0.8- RPE65 (F) into the subretinal space of adult (A and F) or P5 (B-E) mouse retina. A and B show examples of GFPexpressing RPE cells. Dapi counterstaining in A allows visualization of the outer nuclear layer. Arrowhead in C indicates the nucleus of a photoreceptor expressing GFP; note the presence of GFP in the corresponding segment. D shows an example of GFP expression located in cells of the corneal epithelium (E: dapi counterstaining of D). F: immunolabeling of RPE65 in an adult RPE65 knock-out mouse one week after injection of LV-R0.8-RPE65. Expression of the transgenic protein was restricted to RPE cells. G: non-injected knock-out mouse. H: non-injected wild type control. ONL: outer nuclear layer; RPE: retinal pigment epithelium.

confirming previous studies that have demonstrated the high capacity of lentiviral vectors to transduce RPE cells (Miyoshi et al. 1997; Bainbridge et al. 2001; Kostic et al. 2003).

We also performed intravitreal injections of LV-R0.8-GFP into the eye of mouse pups of 5 days of age (P5). This led to high GFP expression in the RPE cells (Fig. 35.2b). Surprisingly, the external surface of the corneal epithelium also expressed a high level of GFP (Fig. 35.2d-e). Although the injection procedure surely resulted in the diffusion of the vector on the eye surface, it was unlikely to observe GFP here with the R0.8 promoter. The reason

for this expression remains unclear, but a transient activation of the RPE65 promoter in these regions cannot be excluded, knowing that RPE65 is expressed in human keratinocytes (Hinterhuber et al. 2004). Occasionally, we also observed some cells expressing GFP in the photoreceptor layer (Fig. 35.2c). The differences in expression pattern between the adult and neonates illustrate the fact that to target a specific cell type, one has to consider the combination of the vector tropism, the injection site, and the specificity of the promoter used. In the present study, the cause of the absence of GFP in photoreceptors after gene transfer in the adult is probably due to an absence of infection of these cells – the vector being known to poorly transduce adult photoreceptors (Kostic et al. 2003). In addition, endogenous RPE65 is not expressed in rods, which represent more than 95% of the mouse photoreceptors, and its expression in cones is unclear (Seeliger et al. 2001; Znoiko et al. 2002).

3.3. Therapeutic Level of RPE65 in the Retina

We next injected the LV-R0.8-RPE65 therapeutic vector into the subretinal space of 2.5 months old RPE65-/- mice. At this age the ERG of these mice is nearly flat (Seeliger et al. 2001). One week after injection, a few mice were sacrificed to assess expression of the transgene. This allowed us to detect significant levels of RPE65 protein in the RPE cells of injected animals (Fig. 35.2f), whereas no RPE65 could be detected in non-injected RPE65-/- mice (Fig. 35.2g). Nevertheless, the level of expression of RPE65 appeared to be weaker in LV-R0.8-RPE65 treated RPE65-/- mice than in wild type controls (compare Fig. 35.2f and 2h).

To assess that the expression of RPE65 was sufficient to reach a therapeutic level, we measured the ERG of the treated animals at three months, i.e. two weeks after injection of the lentiviral vector. In some animals we detected an ERG response characterized by a b-wave amplitude that could reach as much as 50% of wild type controls (Fig. 35.3).

4. CONCLUSION

In the present study we report the first successful attempt to restore visual function in RPE65 knockout mice by injection of a lentivirus encoding RPE65, thus demonstrating that lentiviral vectors could be valuable tools to treat LCA. Moreover, we report that the R0.8 promoter fragment was efficient to drive the expression of therapeutic levels of RPE65 protein. The use of the GFP reporter gene revealed that in the context of a lentiviralmediated gene transfer, R0.8 activity was specific for RPE cells in adult mice. In newborn mice, however, we detected additional expression in the corneal epithelium and in few photoreceptors. Nevertheless, this phenomenon occurred only after injection in neonates, for which (i) the injection procedure is difficult to control and (ii) the differences with the adult extracellular matrix could explain the transduction of photoreceptors.

There is now accumulating body of evidence that RPE65 gene transfer using AAV and the chicken beta-actin promoter in animal models of LCA is beneficial over the long term for visual function (Bennett 2004). For the treatment of human patients, it is nevertheless mandatory to test alternative promoters and vectors in animal models of RPE65 mutations, to achieve safe, sufficient and long-lasting expression of RPE65. Lentiviral vector and RPE65 promoter might provide such an alternative.