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    • The first report of GUCY D related gene augmentation

    2022-05-13

    The first report of GUCY2D-related gene augmentation therapy in mammalians was also reported in 2006 in the KO mouse model (section 4.1.1). In this study, an AAV5 vector by using AAV5-mediated transfer of the bovine cDNA (the same used in the chicken study) was injected to the post-natal KO retina at the age of 3 weeks followed by ERG and immunocytochemical analyses 5 weeks post injection. The treatment effort failed to restore cone ERG responses or prevent cone degeneration but it restored the light-driven translocation of cone arrestin in transduced cone Penciclovir (Haire et al., 2006). In these two studies the injected virus included the bovine Gucy2d sequence (probably since this sequence was historically used in biochemical GC-E functional assays) rather than the species-specific cDNA. To overcome this potential obstacle, AAV5 and AAV8 containing murine Gucy2e were subretinally delivered to the same KO mouse model at P14 and P25 (Boye et al., 2010). Using two promoters (either a photoreceptor specific or a ubiquitous promoter), expression was verified in both rods and cones, leading to long-term improvement in photopic ERG responses (∼45% of normal) lasting for 3 months as well as improved visual behavior (Boye et al., 2010, Boye et al., 2011). The same KO mouse was injected with AAV8 vector containing the human rhodopsin kinase promoter and the human GUCY2D gene (Mihelec et al., 2011). This lead to a long-term effect including an appropriate localization of the transgene product to the outer segments of rods and cones, increased levels and appropriate localization of cone alpha-transducin, higher cone ERG function, improvement in cone-mediated visual function, and a significant improvement in rod ERG. The slightly higher ERG amplitudes seen in the latter study were likely attributed to the earlier treatment time point applied in this study (P10) (Boye et al., 2012). A recent proof of concept experiment used the Nrl(−/−) Gucy2e(−/−) mouse, an all-cone model deficient in GC-E (Boye et al., 2015). AAV5 treatment was used to deliver human GUCY2D to the subretinal space. The treatment fully restored cone function, cone-mediated visual behavior, and guanylate cyclase activity, and preserved cones for at least 6 months. Retinal function could be restored to levels above that in Nrl(−/−) controls, probably due to increased cyclase activity in treated Nrl(−/−) Gucy2e(−/−) mice relative to Nrl(−/−) controls. In addition, delivery of a candidate clinical vector, AAV5-GRK1-GUCY2D, which was shown to be suitable in primate photoreceptors (Boye et al., 2015), restored retinal function that persists for at least 6 months. This study therefore provides strong support for clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1 patients. As discussed in chapter 3.3.1, LCA1 patients exhibit relatively good preservation of retinal structure and slow progression of disease over time, which makes GUCY2D an excellent candidate for gene therapy with a relatively wide window for treatment. Moreover, the successful AAV treatment in the KO mouse model is a proof-of-concept for this treatment modality. In addition, a recent study demonstrated a relatively intact postgeniculate white matter pathway in LCA1 patients, which is encouraging for the prospect of recovery of visual function with gene augmentation therapy (Aguirre et al., 2017). An important aspect of developing gene therapies is to determine the appropriate outcome measures for clinical trials. A recent study (Jacobson et al., 2017) involving 28 LCA1 patients (Ages 2–59 years) showed that conventional measures, such as visual acuity, will need to be complemented by other methods such as chromatic full-field sensitivity testing that provides a photoreceptor-based subjective outcome, and optical coherence tomography to assess photoreceptor structure in the fovea and the superior retina.
    Future perspectives The genetics of GUCY2D-related phenotypes is well-established with a relatively large number of LCA and CRD-causing mutations showing a clear genotype-phenotype correlation. The major advances in understanding GC-E are expected to be achieved by studying the biochemical aspects of GC-E and by developing treatment modalities for LCA1 patients. For a large number of mutations, the scientific community lacks key parameters of GC enzyme function including regulation by affinity for GCAPs and Ca2+sensitive regulation by GCAPs. Using established cell culture techniques for heterologous expression of GC-E will allow the comparison of results from different labs. It should be noted, however, that cell culture analyses lack important parameters that regulate GC-E in vivo. Therefore, parallel studies on transgenic animals (mainly mice) will be needed to explore the effects of mutations in an animal model before the models can be employed for test in gene therapy approaches. It is clear that better understanding of the pathogenicity of GC-E mutations will allow the development of more efficient therapies.