Final Report Summary - AAVEYE (GENE THERAPY FOR INHERITED SEVERE PHOTORECEPTOR DISEASES) Executive Summary:Many blinding disorders are caused by mutations in genes preferentially expressed in the photoreceptor cells of the retina. The objective of the AAVEYE consortium has been to develop state-of-art gene transfer tools for the photoreceptor neurons in the retina, and to provide pre-clinical proof-of-concept of gene therapy for severe blinding retinal photoreceptor diseases. Vectors derived from the adeno-associated virus (AAV) have proven to efficiently transduce the retina of animal models and AAV-mediated gene transfer appears to revert retinal pigment epithelium (RPE) defects. The retina represents an ideal target for gene therapy approaches because of the size of the eye, which allows the use of small vector doses, and because of its immuno-privileged environment. AAVEYE focused, in particular, on two retinal diseases for which no therapies are yet available: Retinitis Pigmentosa (RP) and Leber Congenital Amaurosis (LCA), due to mutations in the PDE6B gene and AIPL1 gene, respectively. The ultimate goal of the AAVEYE consortium has been to develop gene therapy strategies for the treatment of RP and LCA. The safety and feasibility of the AAV-mediated gene transfer strategy for the treatment of Retinitis Pigmentosa and Leber Congenital Amaurosis has been tested by the AAVEYE consortium for the first time, potentially aiming to the phenotypic and functional rescue of inherited severe photoreceptor degenerations.The consortium has intended to accomplish such aim through: 1) the development of AAV-based long-term and safe gene therapy to photoreceptors by combining endogenous promoters and AAV serotypes (WP1); 2) the assessment of the impact of AAV-mediated photoreceptor transduction on rescue of visual function in animal models of severe RP and LCA (WP2); 3) the evaluation of the efficacy of combination of gene replacement with adjuvant molecules on photoreceptor survival (WP3), and 4) the characterization of patients with inherited severe photoreceptor diseases to move the gene therapy strategies tested from bench to bedside (WP4). Project Context and Objectives:Many blinding disorders, for which no therapies are currently available, are due to inherited conditions such as Retinitis Pigmentosa (RP, Weleber and Gregory-Evans 2001) and Leber Congenital Amaurosis (LCA, Weleber and Gregory-Evans 2001) that affect the retina. Over 140 different loci responsible for RP and 10 for LCA (http://www.sph.uth.tmc.edu/Retnet/) have been identified so far. Most of the genes responsible for RP and LCA are expressed in photoreceptors exclusively or in addition to other cell types (Cremers et al. 2002; Weleber and Gregory-Evans 2001). Mutations in many of the genes encoding for proteins involved in phototransduction or in the visual cycle cause inherited retinal degeneration (Weleber and Gregory-Evans 2001). Mutations in PDE6B and AIPL1 are particularly interesting because they are relatively common causes of severe recessively inherited retinal degeneration (PDE6B causes RP and AIPL1 causes LCA). Animal models bearing Pde6b and Aipl1 mutations are available and resemble the disease phenotype observed in humans. bPDE and AIPL1 deficiencies share a common pathogenetic mechanism. PDE6B encodes the β-subunit of the rod-specific phosphodiesterase (bPDE). This protein is an essential element in the phototransduction cascade (Ionita and Pittler 2007). Mutations in the PDE6B gene have long been linked to autosomal recessive retinitis pigmentosa (Bowes et al. 1990; Danciger et al. 1995) as well as autosomal dominant RP [see http://www.retina-international.com/sci-news/pdemut.htm]. Six percent of patients with recessive RP have mutations in PDE6B (Bayes et al. 1996). Two naturally occurring mouse models of disease exist, rd1 and rd10 (Chang et al. 2002), both are due to mutations in the rod-specific Pde6b gene. The AIPL1 gene encodes a photoreceptor cell specific chaperone that is hypothesised to be responsible for correct folding or localisation of the bPDE (Liu et al. 2004; Ramamurthy et al. 2004). Mutations in the AIPL1 gene have been shown to result in Leber’s congenital amaurosis, a form of inherited childhood blindness [see Hameed et al. 2000. The two animal models of disease caused by mutations in the Aipl1 gene are the knockout (Aipl1 -/-) mouse and the hypomorphic mutant (Aipl1 h/h) mouse. In the absence of AIPL1, a lack of functional bPDE results in a disruption of the phototransduction cascade and the subsequent loss of the photoreceptor cells. Mutations in PDE6B and AIPL1 are therefore common causes of recessive severe photoreceptor degeneration in humans and mice through a common mechanism involving bPDE activity. In the case of recessive RP and LCA, supplying a correct copy of the defective gene represents a potential therapeutic strategy for these otherwise untreatable diseases. To this end, vectors derived from the small AAV are the most promising for somatic retinal gene transfer (Surace and Auricchio 2008) and their safety and efficacy was tested for the first time by PARTNER 2 in the retina of patients affected by LCA due to mutations in the gene encoding RPE65 (Bainbridge et al. 2006; Bennett 2006). A single intraocular injection of AAV results in gene expression sustained for years in photoreceptors or RPE of rodents, dogs, cats and non-human primates (Acland et al. 2005; Acland et al. 2001; Ali et al. 1996; Bennett et al. 1999; Ho et al. 2002; Lotery et al. 2003). One of the attractive features of AAV as an in vivo gene transfer vector is that the surface capsid proteins can be easily interchanged among different AAVs thus creating hybrid vectors with the vector genome (encoding the therapeutic gene) from one serotype, i.e. AAV2, and the capsid from a different AAV, i.e. 1 or 5 (the resulting hybrid vectors being named AAV2/1 or 2/5) (Auricchio 2003). Since capsid proteins are the main determinants of AAV tropism and transduction characteristics, vectors with different capsids have different ability to transduce retinal cells in vivo (Auricchio et al. 2001b; Lotery et al. 2003; Rabinowitz and Samulski 2002; Yang et al. 2002). Based on this, PARTNER 1 recently tested the ability of novel AAV serotypes to transduce the murine retina and more specifically the photoreceptors. PARTNER 1 found that AAV2/8 and 2/7 mediate 6- to 8-fold higher in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient for photoreceptor targeting (Allocca et al. 2007). In addition, following subretinal administration of AAV the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested (Allocca et al. 2007). This could be crucial for successful treatment of animal models of photoreceptor diseases, a task more challenging than treatment of RPE-specific retinal defects. AAV2/2, 2/1 and 2/4 have been successfully used to treat RPE specific genetic defects including mucopolysaccharidoses (Hennig et al. 2004; Ho et al. 2002), ocular albinism (Surace et al. 2005), RP and LCA due to MERTK and RPE65 gene deficiencies, respectively (Acland et al. 2005; Acland et al. 2001; Dejneka et al. 2004; Le Meur et al. 2007; Narfstrom et al. 2003; Pang et al. 2006; Smith et al. 2003; Surace et al. 2005). Various centers, including PARTNER 2, are testing the toxicity and tolerability of AAV2/2 vectors in the retina of patients with RPE65 mutations (Bennett 2006). Gene transfer approaches for photoreceptor-specific diseases have been successful in rare cases (Min et al. 2005; Pawlyk et al. 2005; Zeng et al. 2004) probably due to limitations of the vector system, the nature of photoreceptor-specific retinal degenerations and constrains in photoreceptor accessibility by vectors. The ability of AAV vectors to transduce RPE more efficiently than photoreceptors may be related to the simple monolayer organization of the RPE as opposed to the photoreceptors that are higher in number and architectonically organized in rows (Clarke et al. 2000) which may represent a physical barrier to their transduction. The mechanism of photoreceptor loss suggests that gene transfer efficiency may be a limiting factor for photoreceptor rescue. RP initially affects the peripheral retina, resulting in the degeneration of rods, while cones and central vision are preserved at this stage (Dryja 2001). With the progression of the disease, the cones also degenerate (rod–cone degeneration) suggesting a non cell-autonomous mechanism of cell death. Non-autonomous patterns of degeneration are similarly observed in those inherited retinal diseases affecting primarily cones, as cone-rod dystrophies, suggesting a general mechanism of degeneration in inherited retinal diseases (Weleber and Gregory-Evans 2001). Therefore, widespread photoreceptor transduction is desirable to prevent detrimental effects from non-transduced photoreceptors. To this end, the combination of AAV2/8 and the rhodopsin promoter or endogenous promoters may allow successful treatment of severe photoreceptor diseases. Considering the severe nature of some photoreceptor degenerations like those due to bPDE and AIPL1 deficiencies, combinatorial therapies aiming at supplying the correct copy of a mutant gene and at decreasing the progression of photoreceptor degeneration acting on mechanisms downstream of the defective gene could be desirable. The efficacy of several neurotrophic molecules or of small molecule drugs (Frasson et al. 1999; Pawlyk et al. 2005; Pearce-Kelling et al. 2001; Takano et al. 2004; Yamazaki et al. 2002) has been tested in animal models of photoreceptor degeneration. Based on this context, the objectives of the AAVEYE consortium (and the milestones due) have been:1- To optimize AAV-mediated gene transfer to photoreceptors for treatment of bPDE and AIPL1 deficiencies, through combination of AAV serotypes and endogenous promoters.Milestone 1: Levels of bPDE expression in mice with AAV2/8 using endogenous promoters.Milestone 2: Levels of AIPL1 expression in mice with AAV2/8 using endogenous promoters.Milestone 3: Expression level of bPDE in Non Human Primates (NHPs).Milestone 4: Expression levels of AIPL in NHPs.Milestone 5: Feasibility of using endogenous promoters to drive expression of bPDE and AIPL1.2- To test the efficacy of AAV-mediated photoreceptor gene transfer in murine models of Pde6b and Aipl1 gene deficiencies. Milestone 6: Complete production of vectors necessary for gene transfer to murine models.Milestone 7: First report on outcome of photoreceptor survival after gene transfer in models of retinal degeneration.Milestone 8: Final report of efficiency of photoreceptor survival after gene transfer in models of retinal degeneration.3- To combine AAV-mediated gene replacement with molecules prolonging photoreceptor survival, like the neurotrophic factor GDNF, Ca++ channel blockers and RNA interference of the polycomb protein Bmi1.Milestone 9: Report on tumor suppressor action on photoreceptor survival.Milestone 10: Long-term photoreceptor survival by combined gene replacement therapy with GDNF.Milestone 11: Long-term photoreceptor survival induced by combined gene replacement therapy with Nilvadipine.Milestone 12: Synergism between Bmi1 inhibition or Rb treatment and PDE6B/AIPL1 gene transfer.Milestone 13: Report on micro-array analysis to identify survival pathways.4- To characterize at the molecular and clinical levels RP and LCA patients to identify candidates to move AAV-mediated PDE6B and AIPL1 gene transfer from bench to bedside.Milestone 14: Molecular characterization of patients available from beginning of project.Milestone 15: Report of clinical characterization of PDE6B patients, the optimal time interval for gene therapy and the recommended clinical tests before gene therapy.Milestone 16: Report on the clinical characterisation of AIPL1 patients, the optimal time interval for gene therapy and the recommended clinical tests before gene therapy.Project Results:During the AAVEYE granting period the PARTNERS have achieved the following objectives:Workpackage 1 Optimization of AAV-mediated photoreceptor gene transfer for gene therapy of bPDE and AIPL1 deficienciesObjectivesThe goal of WP1 was to obtain AAV-mediated photoreceptor gene transfer that could mimic physiological levels of bPDE and AIPL1 expression. For this purpose, the PARTNERS used AAV2/8 vectors (that have the highest levels of photoreceptor gene transfer among a series of AAV serotypes tested, Allocca et al. 2007) in combination with fragments of the endogenous PDE6B and AIPL1 5’ regulatory regions, based on the assumption that endogenous promoters should reproduce more faithfully the expression levels and timing of a transgene than exogenous regulatory regions. Description of workThe viral vector based on adeno-associated virus serotype 8 (AAV2/8) has been identified as the most efficient to transfer genes to photoreceptors (PR). The PARTNERS demonstrated that this AAV2/8 ability is retained across various species (mice and pigs). This is a major achievement in the field as: i) diseases requiring gene transfer to PR are a major cause of blindness; ii) the vectors available before our discovery were unsatisfying in terms of rescue of PR-specific diseases in animal models. In addition, PARTNER 1 described a strategy to tailor tightly and efficiently therapeutic gene expression to PR based on exploiting small non-coding RNAs (miRNAs), which are physiologically expressed in retinal PR cells. The results from these studies pertain to WP1 and are reported in the following publications: Karali et al., 2011; Mussolino et al., 2011 (see PDFs attached)As there was a report that the vector could spread outside the eye, PARTNER 2 looked at the presence of gene transfer to the brain. Six dogs (4 young, 2 adults) were injected subretinally with AAV2/8 vectors carrying a marker gene under control of a ubiquitous promoter. Examination of the retina showed widespread expression of the transgene, as expected for this vector. There was evidence of limited vector spread outside the eye, with transgene expression apparent in the optic nerve and optic chiasm and sporadic expression in the brain. Therefore, the vector system can effectively transfer genes to the photoreceptor cells in rodents and large animals. Moreover, it appeared that spread of the vector to the brain is not a major problem and is unlikely to substantially affect the feasibility of retinal gene therapy using AAV2/8 vectors.In parallel to the demonstration that AAV2/8 is an efficient vector for gene transfer in the retina of models of severe retinal degeneration, the PARTNERS tested different sequences of the PDE6b promoter in order to drive efficient PDE6b expression specifically in photoreceptors. The promoter sequence had been previously characterized by the group of Farber (Di Polo and Farber, 1995; Di Polo et al. 1997), but not for gene transfer purposes. PARTNERS 3 identified a sequence that allows inducing high and rapid expression of the transgene. This rod-specific promoter sequence is the shortest (149 bp) allowing efficient gene transfer and is thus of great interest for AAV vector design in general since it gives more space for the transgene of interest and good expression.In conclusion, the PARTNERS:- demonstrated that the viral vector based on adeno-associated virus serotype 8 (AAV2/8) is the most efficient to transfer genes to photoreceptors;- demonstrated that the AAV2/8 vector system can effectively transfer genes to the photoreceptor cells in rodents and large animals;- described a strategy to tailor tightly and efficiently therapeutic gene expression to PR based on exploiting small non-coding RNAs (miRNAs), which are physiologically expressed in retinal PR cells;- reported that spread of the vector to the brain is not a problem and is unlikely to substantially affect the feasibility of retinal gene therapy using AAV2/8 vectors;- identified a rod-specific promoter sequence that allows inducing high and rapid expression of the transgene, allowing efficient gene transfer.WP2Gene replacement therapy in Pde6B and Aipl1 murine mutantsObjectivesThe overall goal of WP2 was to test the efficacy of state-of-the-art AAV-mediated photoreceptor gene transfer in animal models of Pde6b and Aipl1 deficiencies as well as providing proof-of-principle of gene replacement therapy for severe photoreceptor diseases due to PDE6B and AIPL1 mutations.Description of workThe PARTNERS showed that subretinal administrations of AAV2/8, which has high PR gene transfer ability, results in morphological PR protection in animal models of Retinitis Pigmentosa and Leber congenital amaurosis type 4 (LCA4). Indeed, PARTNER 1 observed that neonatal subretinal injections of AAV2/8 carrying the correct copy of the genes mutated in the two forms of inherited retinal degenerations (PDE6B mutated in RP and AIPL1 mutated in LCA4) significantly slowed the fast PR degeneration present in the retinae of these two murine models. The data produced by PARTNER 1 are contained in the following publications: Allocca et al., 2011; Testa et al., 2011 (see PDFs attached).For the treatment of Leber congenital amaurosis type 4, PARTNER 2 produced AAV2/8 vectors carrying the AIPL1 gene. After subretinal administration of AAV2/8.CMV.AIPL1 in Aipl1 knock-out mice, which have an extremely rapid retinal degeneration, there was production of AIPL1 protein, an improvement in retinal function and a survival of photoreceptor cells for at least 6 weeks post-treatment, which is about 4 times longer than the natural photoreceptor survival in these animals. The AAV2/8.RK.AIPL1 construct that drives AIPL1 expression in the photoreceptors only, was tested in a manner similar to the study above. PARTNER 2 could show in Aipl1-/- mice that this vector supplemented the AIPL1 and restored the PDE production and localisation. There was a rescue of retinal function and a prolongation of photoreceptor survival for up to 5 months. As photoreceptor specific expression of the transgene adds to the safety aspects of a novel gene therapy vector, PARTNER 2 has chosen to use the AAV2/8.RK.AIPL1 vector in a planned clinical trial testing the safety and efficacy of AAV2/8-mediated gene therapy for LCA4. The results of the AIPL1 gene replacement studies by PARTNER 2 are published in Tan et al. (2009) and Sun et al. (2010) (see PDFs attached). An AAV2/8 vector carrying the therapeutic transgene controlled by the PDE6b promoter was tested by PARTNER 3 for restoring gene function in a severe form of an animal model of retinitis pigmentosa, the Rd10 mouse which presents defective Pde6b expression. PARTNER 3 observed that the gene therapy approach allows correcting the expression of the missing protein, rescuing a large number of photoreceptors and, more importantly, restoring retina function. Such rescue can be achieved when the vector was administrated just before the initiation of cell death, when the photoreceptors start to degenerate (inner segments already reduced) indicating that this therapeutic approach may be efficient in human even with altered photoreceptors.In conclusion the PARTNERS demonstrated that:- administration of AAV2/8 carrying the correct copy of the genes mutated (PDE6B mutated in RP and AIPL1 mutated in LCA4) significantly slows down the fast photoreceptors degeneration and restores retina function. Workpackage 3Combined therapies to restore vision in Pde6b and Aipl1 murine mutants.ObjectivesWP1 and WP2 aimed to develop a clinically relevant viral vector to efficiently target photoreceptors. In the case that gene transfer, in the most severe forms of Pde6b and Aipl1 deficiency, is not be sufficient to markedly delay photoreceptor death, combined strategies were envisaged using gene transfer of the wild-type gene together with treatment with additional components such as the neurotrophic factor GDNF, calcium blockers, or activation of tumor suppressors controlling photoreceptor differentiation.Goal of this workpackage was to test the potential synergistic effect of neurotrophic molecules, Ca++-channel blockers and cell-cycle manipulation with gene replacement on photoreceptor survival in Pde6B and Aipl1 deficient models.Description of workIn order to improve the therapeutic efficacy obtained by gene therapy in the rd10 mouse model of retinitis pigmentosa, the PARTNERS coupled to subretinal administrations of AAV2/8 methods to slow down retinal degeneration such as: i) dark-rearing; ii) systemic administrations of calcium inhibitors; iii) intraocular gene delivery of a neurotrophic factor. All these strategies are known to slow PR loss either in this (i and ii) or in other rodent models of PR degeneration (iii). Unfortunately, none of these combinations resulted in higher therapeutic efficacy than gene therapy alone. Thus, the consortium proposed intraocular AAV2/8-mediated delivery of the correct copy of the PDE6B gene mutated in this form of RP as the gene therapy strategy of choice. These results pertain to WP3 and are contained in the following publication: Allocca et al., 2011 (see PDF attached).To develop a gene therapy protocol for RP40, PARTNER 2 injected AAV2/8 vectors carrying the PDE6B gene subretinally into mouse models of disease. Treatment resulted in a modest replacement of PDE protein in the treated, but not the untreated eyes of the PDE deficient mice. However, this did not lead to a significant improvement of photoreceptor survival. Further development of the treatment strategy using neurotrophics or small molecule inhibitors of the ion channels did not yield a substantial improvement of the therapeutic effect. However, preliminary results suggested that genetic inhibition of the calcium channels may prolong the survival of the photoreceptors and rescue the photoreceptors and this line of research is continuing.In parallel to these studies, PARTNER 3 identified a new cellular pathway that leads to photoreceptor death. PARTNER 3 observed that several cell cycle proteins are expressed before the state of photoreceptor death. Using genetic models or drug testing in vitro on cultured retina explants, PARTNER 3 showed that the inhibition of certain cell cycle proteins rescues transiently or markedly both rods and cones depending on the molecular target. Different vectors were then produced in order to attempt to delay photoreceptor death during retinal degeneration of the Rd10 mouse by interfering with some cell cycle protein functions. None of these vectors showed efficient rescue even when combined with the therapeutic AAV2/8- PDE6b vector. However, a pharmacological approach showed promising results in vitro by inhibiting Cdks, but several developments are still necessary to be performed to deliver efficiently this compound into the eye during a long period. In summary, these series of experiments clearly show that cell cycle proteins play a major role in the execution of the photoreceptor death process in several models of retinal degeneration. Moreover, Cdk proteins appear to be a realistic potential therapeutic target, whereas Bmi1 protein, which was thought at the beginning to be a valid candidate, appears difficult to control. This is probably because Bmi1 participates to histone and DNA modifications, which are also necessary to re-arrange in order to restore a normal gene expression pattern. In parallel PARTNER 3 identified that Bmi1 prevents Müller cells from regenerating the photoreceptor layer. In the absence of Bmi1, Müller cells can de-differentiate and migrate to the photoreceptor layer, their final fate remaining to be determined. This observation also opens a new field of investigation to manipulate these cells via Bmi1 to generate new photoreceptors. Of course, for genetic diseases, an adequate gene replacement strategy remains to be undertaken to restore a visual function. The vectors developed in the present study show the feasibility of using the AAV2/8 vector with a specific photoreceptor promoter for restoring gene function in a severe form of retinitis pigmentosa. These gene therapy results consolidate similar observations obtained recently with the same vector in various models, including large animals, rendering this vector a good candidate for human applications.A manuscript entitled “Retinal degeneration depends on Bmi1 function and re-initiation of cell cycle machinery” Zencak, D., Chen, D., Ekström, P., Tanger, E., Bremner, R., van Lohuizen M., Arsenijevic, Y., is in revision (submitted to PNAS). In conclusion, in this workpackage the PARTNERS demonstated that:- treatment strategy using neurotrophic factors or small molecule inhibitors of the ion channels does not yield a substantial improvement of the gene therapy therapeutic effect, therefore intraocular AAV2/8-mediated delivery of the correct copy of the gene mutated is the gene therapy strategy of choice.In addition,- a new cellular pathway that leads to photoreceptor death has been identified- a gene implicated in preventing retinal regeneration has been identified.Workpackage 4Molecular and clinical characterisation of RP/LCA patients with PDE6B and AIPL1 mutations.ObjectivesIn view of future potential applications of gene therapy, the AAVEYE consortium performed the molecular and clinical characterization of RP/LCA patients with PDE6B and AIPL1 mutations.Description of workIn view of future potential applications of gene therapy, the AAVEYE consortium performed the molecular and clinical characterization of RP/LCA patients with PDE6B and AIPL1 mutations.PARTNER 1 characterized at the molecular level series of patients with sporadic or recessive RP as well as LCA who were followed up at the Second University of Naples Department of Ophthalmology. PARTNER 1 identified 10 LCA patients with mutations in the AIPL1 gene, thus then defined as LCA4 patients, as well as 4 RP patients carrying mutations on both PDE6B alleles. The patients with PDE6B mutations presented with typical RP and severe PR degeneration throughout the retina, according to a rod-dominated disorder. The patients with LCA4 had early onset severe retinal dystrophy with marked macular degeneration. However in most of the Italian LCA4 patients including older individuals, perifoveal PR appears to be spared by the degenerative process suggesting that these PR can be the target of gene therapy. PARTNER 1 results on the LCA4 patients have been published in Testa et al., 2011 (PDF attached).Approximately 400 DNA samples from the early-onset severe retinal degeneration patient database of Moorfields eye hospital were screened (by PARTNER 2) for mutations in a range of retinal dystrophy genes. PARTNER 2 was able to identify seven LCA patients with mutations in AIPL1 that are likely to be disease-causing, but no patients with PDE6B mutations were identified. Direct sequencing led to the identification of 29 novel AIPL1 sequence variants, and computer analysis suggests that 6 of these variants are likely to be disease-causing mutations. The ages of identified AIPL1-deficient patients ranged from 2 years to 36 years. Although the LCA phenotype is characterised by early onset severe visual loss in our patient series, some of the retinal structure and retinal function appeared to be preserved in some of the patients. This phenomenon was most marked in the youngest patient in our cohort, who had a measurable visual acuity. In this subject assessment of electrophysiology revealed reduced cone photoreceptor function, but rod responses were within normal limits. These data suggested that there are patients who have a reasonable window of opportunity for LCA4 gene therapy in childhood. A manuscript describing the characterisation of the AIPL1-deficient patients at Moorfields Eye Hospital is submitted for publication (Tan et al., 2011).To evaluate whether a gene therapy approach can be proposed to patients affected by the PDE6b mutations, PARTNER 3 screened RP and LCA patients in Switzerland. No individuals were identified for PDE6b mutations, but one LCA4 patient was detected. PARTNER 3 enlarged the patient cohort by including families from Tunisia with the collaboration of the Service of Ophthalmology in Tunis (Prof. L. El Matri) and identified 2 patients with the same mutation, and 6 showing only one variant. The characterization of the patient phenotype revealed a massive alteration of the retina with the presence of cysts/oedemas and vitreous attachment leading to retina detachment. Gene therapy for these patients appears very difficult if the therapy is not undertaken at a very early age, and by targeting a large portion of the retina. Indeed, if the retina is only treated locally, we can expect that the cyst formation in the non-treated area with retina attachment on the vitreous will deteriorate the treated region. It thus appears that PDE6b patients are not eligible for a first gene therapy clinical trial with the AAV2/8 vector to show the safety and (some) efficacy of the treatment.In conclusion, panels of DNA samples from patients diagnosed with severe, early-onset retinitis pigmentosa and Leber’s congenital amaurosis, available from the Eye Hospitals in London, Naples and Lausanne, have been screened at ASPER Biotech, Tartu, Estonia (PARTNER 4). 575 DNA samples have been screened during the project for mutations in the PDE6B or the AIPL1 genes using the arRP gene chip or the LCA gene chip, respectively. The arRP gene chip tests for the presence of 594 known disease-associated sequence variants in any of 18 genes, including 26 sequence variants in PDE6B. The LCA gene chip tests for the presence of 641 known disease-associated sequence variants in any of 13 genes, including 40 sequence variants in AIPL1. Mutations in PDE6B and AIPL1 identified using the gene chips were confirmed with direct sequencing by other PARTNERS. The analysis results showed that a PDE6B mutation has been identified in ca 2 % of DNA samples tested for arRP chip (5 DNAs out of 278) and an AIPL1 mutation has been identified in ca 4% of DNA samples tested for LCA chip (11 DNAs out of 283). The arRP gene chip tests for the presence of 594 known disease-associated sequence variants in any of 18 genes, including 26 sequence variants in PDE6B. From 278 arRP patient DNAs, 116 mutations in 13 genes were identified. 4 mutations in five different DNAs were identified in PDE6B gene. A table, describing arRP patient DNA genotypes is included as a Table 1. The LCA gene chip tests for the presence of 641 known disease-associated sequence variants in any of 13 genes, including 40 sequence variants in AIPL1. From 283 LCA patient DNAs, 115 sequence variants from 13 genes were identified. 6 different mutations in eleven DNAs were identified in AIPL1 gene. A table, describing LCA patient DNA genotypes is included as a Table 2.Potential Impact:Discoveries typically begin at “the bench” with basic research — in which scientists study disease at a molecular or cellular level — then progress to the clinical level, or the patient's “bedside.” The AAVEYE proposal has been an example of this type of research; the results obtained by this proposal could have an immediate impact on patient health, by using the preclinical data obtained in the animal models as starting point for the development of clinical trials. Therefore, the consortium has established collaborations with important clinical researchers in each Partner state to provide patients, to assess clinical phenotypes, in order to determine markers and clinical progression that will be fundamental knowledge to evaluate the efficacy of a future therapy.The PARTNERS already had close associations with patient organizations (eg PARTNER 1: Retina Italia, Italian Association Leber's Congenital Amaurosis; PARTNER 2: British Retinitis Pigmentosa Society, Foundation Fighting Blindness; and PARTNER 3: Swiss patient association Retina Suisse), this collaboration with patients association allowed the consortium to rapidly disseminate results to patient organizations, to raise awareness of the state-of–art of new therapeutic approaches and to recruit new patients for molecular analysis and future clinical trials.The potential of the results emerging from this proposal could impact:1) the design of novel AAV vectors and the application of this vector system for the treatment of diseases that affect the photoreceptor cells of the retina. 2) the development of combinatorial therapies for severe photoreceptor diseases by testing alternative strategies with growth factors in order to increase therapeutic window and potential. The results from this objective will open new perspectives for the use of combinatorial therapies summing the effect of gene replacement with adjuvant molecules for these as well as other CNS diseases.3) the possibility to properly treat millions of patients suffering severe retinal degenerations due to photoreceptor demise. AAVEYE consortium have paved the way for future gene therapy trials in humans for severe retinal degenerations by studying the clinical outcome of photoreceptor degenerations in humans and identifying target patients for the gene therapy strategies selected.4) the fine tuning of therapies for diseases affecting other parts of the central nervous system, being the retina an accessible part of the CNS and given the similarities in viralvector transduction between the retina and the CNS.The scientific results of the Project have been disseminated as follows:- Data have been discussed in lab meetings, conference calls, closed meetings and open conferences. - Data have been used to generate hypothesis for further research, for diagnostic possibilities, and therapeutic potential.- Data have been published in the scientific literature.- Results of the project have also been presented in international conferences and meetings. - A public Website has been developed and is online (www.aaveye.eu). Scope of the website is two-fold: to favour dissemination of the project to the scientific community and lay public, and to facilitate inter-communication among PARTNERS and accelerate work progression. This site provides information concerning the objectives, participants, achievements, publications, meetings, employment opportunities and other pertinent information regarding the overall project. The target audience will not only be academic researchers, but also patient groups, industry, biotechnology, and training institutions.To this end, the site consists of a public area and a private area that is password protected (username: PARTNERS; password: europa4). In the public area, which is easily accessible for any internet user, a first section (What is AAVEYE?) provides general information on the focus of the project, the diseases of interest to the consortium, and the aims and impact of the project in terms of EU scientific and societal objectives. A more specific description of the project workpackages and their objectives is also provided together with a general overview of each partner making up the consortium and his/her role in the project, the key personnel and publications acknowledging AAVEYE. Links to other websites, relevant events and participation to meetings and symposia is also available.Finally, a separate section (Internal Use) with sensitive data (such as the TA, the contract and terms) is restricted to the PARTNERS of the consortium and is password protected.The AAVEYE management team has also given particular attention to keep the website up to date with the consortium annual meetings, including the PPTs presented by each partner and Minutes of each meeting, and with the publications acknowledging AAVEYE. The PPTs, Minutes of the meetings and full articles of AAVEYE publications are again restricted to the PARTNERS and the EU and therefore are password protected.Communication within the consortium flowed steadily. Participants have frequently been in contact with each other, via email or skype, to exchange ideas, discuss about the advancement of research and guarantee that it was focused towards the objectives, and to make strategic decisions (when necessary) regarding scientific planning and financial aspects of the programme.Exchange among PARTNERS has also been facilitated by the fact that PARTNERS have been invited to the respective research institutes to give formal lectures. As an example, PARTNER 3 gave a seminar at UCL (PARTNER 2) in June 2009. Also PARTNER 1 was invited at UCL in November 2009 and gave a seminar entitled “Gene therapy for inherited retinal dystrophies: from viral vector to patient”. In February 2010, a senior Research Fellow of PARTNER 2’s laboratory was invited to Lausanne (PARTNER 3) to discuss data on retinal gene therapy in the lab and the clinic in a public seminar. In addition, PARTNER 2 gave a seminar at TIGEM (PARTNER 1) in June 2011, entitled “Development of photoreceptor transplantation strategies for the treatment of retinal degeneration”.Moreover, PARTNERS 1 and 2 have cooperated in another EU project (CLINIGENE-NoE). The aim of the CLINIGENE consortium, consisting of 38 PARTNERS, was to create a European network (EC-NoE) for the advancement of clinical gene transfer and therapy by fostering the interaction of all stakeholders in the field in order to facilitate and help harmonize ethical, quality, safety, efficacy and regulatory issues.In addition, PARTNERS 2 and 3 have both participated to the Retina Suisse Conference in Fribourg (Switzerland) in June 2009 and to the documentary film Malvoyants: lueurs d’espoir, broadcasted on the Télévision Suisse Romande (see sections Seminars and Conference Attendance and Science Dissemination and the Media for major details), where they both also contributed to the design, other than the science, of the documentary film. Powiązane dokumenty final1-223445-1097086-aaveye-finalreport.pdf
