Final Report Summary - CRUMBS IN SIGHT (Restoring Mueller glia cell photoreceptor interactions with Crumbs)
Executive summary:
Retinitis pigmentosa (RP) denotes a clinically and genetically heterogeneous group of hereditary retinal degenerations. Typical RP begins already early in life with night blindness and a progressive loss of peripheral vision due to successive degeneration of rod photoreceptors. The subsequent degeneration of cone photoreceptors at more progressed stages of RP (which often are reached at quite young ages) results in the loss of useful central vision and blindness. Although approximately 1 in 3000 people is affected by RP and allied disorders, no drugs or therapeutics against these diseases are currently available.
The Crumbs homolog 1 (CRB1) gene is mutated in progressive types of RP, in Leber congenital amaurosis (LCA) and in RP with Coat's-like exsudative vasculopathy. In progressive RP due to autosomal recessive mutations in the CRB1 gene, patients experience night blindness from early childhood and a progressive loss of visual field starting in the second decade of life. The quality of life of LCA patients is even more affected, since they are generally born blind or lose their sight shortly after birth. The CRB1 protein shows high homology to the Crumbs protein previously identified in the fruit-fly Drosophila melanogaster. The Crumbs trans-membrane protein acts as a key player in epithelial polarisation and cell-cell adhesion, prevents degeneration of photoreceptors. Similarly, in human and mouse retinas, CRB1 is required to prevent loss of adhesion between Muller glia cells and photoreceptors, pointing to essential interactions between these two cell types for survival of photoreceptor cells. CRB1 is localised specifically in Muller glia cells. The CRB2 and CRB3 proteins are likely to have partially redundant functions in the retina with CRB1.
The consortium used convenient neurobiological model systems (Muller glia cells and photoreceptors; fly photoreceptor cells) that enabled us to gain insight into neuron-glia interactions in general. We determined the cellular effects of over-expression, mis-expression, loss-of-expression and silencing of other CRB-interacting proteins (MPP5/PALS1, MPP3, MUPP1 and PATJ) in primary retina cultures, polarised cell lines, and Drosophila tissues using endogenous cell polarity markers and functional assays. We investigated the redundancy in CRB1 and CRB2 function, and MPP5 and MPP3 function, in Muller glia H photoreceptor interactions, the precise time of onset of the disease, and the cellular and molecular changes occurring during the onset of the disease. We explored the use of Muller glia progenitor cells for retinal transplantation, and setup adeno-associated virus (AAV)-based gene therapy for the transduction of Muller glia cells, and optimised the production of AAV hCRB1 gene therapy vectors for future clinical use.
We conclude that fruit fly Crumbs prevents photoreceptor cell degeneration by stabilising an unconventional Myosin, MyoV, and hence contributes to proper Rhodopsin transport. In cultured cells, there is a post-translational balance between PALS1, PATJ and MUPP1. Mouse CRB2, MPP5/PALS1 and MPP3 play together with CRB1 important roles in neuron-glia interactions in the retina. Mouse CRB1 and CRB2 regulate the suppression of proliferation of late born retinal progenitors, and loss of both CRB1 and CRB2 result in overgrowth of retina tissue. Muller glia progenitor cells have potential use for retinal transplantation. We developed AAV hCRB1 gene therapy vectors to strengthen Muller glia cell photoreceptor interactions, and which will be further developed for clinical use.
Project Context and Objectives:
Retinitis pigmentosa (RP) denotes a clinically and genetically heterogeneous group of hereditary retinal degenerations. Typical RP begins already early in life with night blindness and a progressive loss of peripheral vision due to successive degeneration of rod photoreceptors. The subsequent degeneration of cone photoreceptors at more progressed stages of RP (which often are reached at quite young ages) results in the loss of useful central vision and blindness. Although approximately 1 in 3000 people is affected by RP and allied disorders, no drugs or therapeutics against these diseases are currently available.
The Crumbs homolog 1 (CRB1) gene is mutated in progressive types of RP, in Leber congenital amaurosis (LCA) and in RP with Coat's-like exsudative vasculopathy. In progressive RP due to autosomal recessive mutations in the CRB1 gene, patients experience night blindness from early childhood and a progressive loss of visual field starting in the second decade of life. The quality of life of LCA patients is even more affected, since they are generally born blind or lose their sight shortly after birth. Immunohistochemical studies on retinas revealed that the mouse and human CRB1 protein is exclusively localised at so-called subapical regions (SARs) adjacent to adherens junctions (AJs) between Muller glia (MG) cells and photoreceptors (PRs) at the outer limiting membrane (OLM). Immuno-electron microscopy on mouse retinas furthermore revealed that CRB1 protein is exclusively present at the SAR of Muller glia cells, whereas CRB family members CRB2 and CRB3 and CRB-interacting proteins localise at the SAR of Muller glia cells and photoreceptors.
The CRB1 protein shows high homology to the Crumbs protein previously identified in the fruitfly Drosophila melanogaster. The Crumbs transmembrane protein acts as a key player in epithelial polarisation and cell-cell adhesion, and prevents degeneration of photoreceptors. Similarly, in human and mouse retinas, CRB1 is required to prevent loss of adhesion between Muller glia cells and photoreceptors, pointing to essential interactions between these two cell types for survival of photoreceptor cells. The CRB2 and CRB3 proteins are likely to have partially redundant functions in the retina with CRB1.
The consortium uses a convenient neurobiological model system (Muller glia cells and photoreceptors; fly photoreceptor cells) that enables us to gain insight into neuron-glia interactions in general. We have integrated high quality expertise that is available across Europe in order to unravel the biochemical and pathophysiological pathways that lead to retinal degeneration in progressive RP and LCA. With the knowledge obtained in this project it will be possible to devise and optimise novel therapies, which will already commence within the timeframe of this project.
It is the overall aim of the CRUMBS IN SIGHT consortium to decipher the function of CRB1 in Muller glia cell photoreceptor interactions, and to develop therapies to restore Muller glia cell photoreceptor interactions mediated by CRB1 in the visual system. The EC proposal can be divided into the following five key objectives addressed in five Work Packages (WP1-WP5):
WP1) Determination of the cellular function of Drosophila, mouse, and human CRB and CRB interacting-proteins (CIPs) in polarised cells and prevention of photoreceptor death.
WP2) Studying the primary defects leading to loss of Muller glia cell photoreceptor interaction in conditional Crb2 knockout retinas and embryos.
WP3) Studying the primary defects leading to loss of Muller glia cell photoreceptor interaction in conditional Pals1 gene silenced (knockdown) retinas and embryos.
WP4) Development of Muller glia progenitor cell transplantation.
WP5) Optimisation of AAV6 capsids for specific transduction of Muller glia cells, and optimisation of AAV2/6 hCRB1 clinical gene therapy vector production.
The proposed experiments are complementary to an ongoing viral hCRB1 gene therapy programme at the Netherlands Institute for Neuroscience. Our expectations are the following: We expect to obtain insight in the function of the CRB complex in regulation of Muller glia cell photoreceptor interaction. We expect to develop safe and efficient therapeutic strategies for cures for neurosensory eye disorders. Our approaches are the following:
WP1 will determine the cellular function of Drosophila, mouse, and human CRB and CRB interacting-proteins (CIPs) in polarised cells and prevention of photoreceptor death. Recent evidence indicated that the CRB-interacting protein PALS1 (MPP5; Stardust) is required for correct localisation of CRB, CRB1, CRB2, and CRB3 as well as several other CRB-interacting proteins at the SAR in mammalian retina and the stalk membrane in adult Drosophila photoreceptors. PALS1 is a member of the membrane associated guanylate kinase- (MAGUK) family of scaffolding proteins. The cellular effects of over-expression, mis-expression and silencing of other CRB-interacting proteins will be evaluated in primary retina cultures, polarised cell lines, and Drosophila tissues using endogenous cell polarity markers and functional assays.
WP2 will analyze retinas and embryos lacking a functional Crb2 gene. Recently we successfully generated and analyzed two Crb1 mutants, and showed an important function for CRB1 in Muller glia cell photoreceptor interactions. CRB1 colocalises with CRB2 and CRB3 at the SAR of Muller glia cells. As recent evidence indicated an important regulatory role for CRB proteins in the formation of tight junctions adjacent to adherens junctions in polarised epithelial cell lines, we will generate conditional Crb2 knockout mice. The mouse model shall help us to investigate redundancy in CRB function in Muller glia cell photoreceptor interactions, the precise time of onset of the disease, and the cellular and molecular changes occurring during the onset of the disease. Crb1/Crb2 double knockout mice will be used to test the efficacy of viral CRB1 gene therapy vectors generated in WP5.
WP3 focuses on the analysis of retinas and embryos lacking a functional Pals1 gene. Previously, we showed by Pals1 gene silencing that PALS1 is required for correct localisation of CRB1, CRB2, CRB3, MUPP1 and VELI3 at the SAR adjacent to adherens junctions at the outer limiting membrane in primary cultured retinas. However, cells that lacked PALS1 still showed morphologically normal Muller glia cell photoreceptor interactions. As it is conceivable that a correct localisation of the CRB1 complex at the SAR is needed for regular functionality, we will generate conditional Pals1 silenced mice. The mouse model shall help us to understand the function of the CRB complex in Muller glia cell photoreceptor interactions, and the cellular and molecular changes occurring during the onset of the disease.
WP4 will study establishment and maintenance of Muller glia cell photoreceptor interaction after subretinal injection of Muller glia cell progenitor cells, and will develop Muller glia progenitor cell therapy. Recently, one of our collaborators showed efficient photoreceptor progenitor transplantation and increased synaptic activity in the mouse eye. A photoreceptor transplantation strategy will not work in patients with mutations in the CRB1 gene because the origin of the defect resides in the Muller glia cells. We are capable of purifying Muller glia cells expressing GFP from different retinal stages, and inject them into the subretinal space. We will determine the optimal differentiation stage of Muller glia cells to be used for transplantation, and study the interaction of the GFP-tagged Muller glia cell with the photoreceptors. In rescue studies, we will investigate the efficacy of this approach to prevent or delay the onset of retinal degeneration in Crb1 mutant mice, particularly with regard to a potential future application in patients with the corresponding neurosensory eye disease.
WP5 will setup adeno-associated virus (AAV)-based gene therapy for the transduction of Muller glia cells, and will optimise the production of clinical AAV2/6 hCRB1 gene therapy vectors. Recently, we identified an AAV serotype capable of efficiently transducing Muller glia cells when intravitreally injected. We will use molecular evolution techniques on the AAV6 capsid to generate AAV serotypes capable of specifically transducing Muller glia cells. Recently, we developed a baculovirus production platform for the AAV1 serotype that is now used for production of clinical (GMP) grade vector. We will develop a similar platform for the AAV6 serotype. We expect to develop therapies (Muller glia stem cell transplantation and AAV CRB1 gene therapy) against progressive RP and LCA that may be used or further developed for clinical trials.
The CRUMBS IN SIGHT project is of vital importance to generate novel therapies against retinal degeneration. The knowledge obtained about the Muller glia cell photoreceptor interaction, and the generated materials and protocols in Muller glia cell transplantation and AAV-based CRB1 gene therapy will be patented, published and disseminated by the consortium. The results will be exploited by technology transfer and by starting a demonstration project in collaboration with a small-medium enterprise.
Project results:
(1) Foreground obtained from work package 1, the determination of the cellular function of Drosophila, mouse, and human CRB and CRB interacting-proteins (CIPs) in polarised cells and prevention of photoreceptor death:
Morphogenesis of Drosophila photoreceptor cells includes the subdivision of the apical membrane into the photosensitive rhabdomere and the associated stalk membrane, as well as a considerable elongation of the cell. Drosophila Crumbs (Crb), an evolutionarily conserved transmembrane protein, organises an apical protein scaffold, which is required for elongation of the photoreceptor cell and extension of the stalk membrane. To further elucidate the role played by different Crb domains during eye morphogenesis, we performed a structure-function analysis in the eye. The analysis showed that the three variants tested, namely full-length Crb, the membrane-bound intracellular domain and the extracellular domain were able to rescue the elongation defects of crb mutant rhabdomeres. However, only full-length Crb and the membrane-bound intracellular domain could partially restore the length of the stalk membrane, while the extracellular domain failed to do so. This failure was associated with the inability of the extracellular domain to recruit beta(Heavy)-spectrin to the stalk membrane. These results highlight the functional importance of the extracellular domain of Crb in the Drosophila eye. They are in line with previous observations, which showed that mutations in the extracellular domain of human CRB1 are associated with retinitis pigmentosa 12 and Leber congenital amaurosis, two severe forms of retinal dystrophy (Richard et al., 2009, A role for the extracellular domain of Crumbs in morphogenesis of Drosophila photoreceptor cells, Eur J Cell Biol. 88:765-77).
Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organise supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organisation is ideally suited to organise protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localised at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis (Bulgakova et al., 2010, Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells, Mol Biol Cell. 21:3915-25).
The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localisation. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localisation of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localisation. We also show a novel function for retromer in maintaining epithelial cell polarity (Pocha et al., 2011, Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs, Curr Biol. 21:1111-7).
Photoreceptor morphogenesis in Drosophila requires remodeling of apico-basal polarity and adherens junctions (AJs), and includes cell shape changes, as well as differentiation and expansion of the apical membrane. The evolutionarily conserved transmembrane protein Crumbs (Crb) organises an apical membrane-associated protein complex that controls photoreceptor morphogenesis. Expression of the small cytoplasmic domain of Crb in crb mutant photoreceptor cells (PRCs) rescues the crb mutant phenotype to the same extent as the full-length protein. Here, we show that over-expression of the membrane-tethered cytoplasmic domain of Crb in otherwise wild-type photoreceptor cells has major effects on polarity and morphogenesis. Whereas early expression causes severe abnormalities in apico-basal polarity and ommatidial integrity, expression at later stages affects the shape and positioning of AJs. This result supports the importance of Crb for junctional remodeling during morphogenetic changes. The most pronounced phenotype observed upon early expression is the formation of ectopic apical membrane domains, which often develop into a complete second apical pole, including ectopic AJs. Induction of this phenotype requires members of the Par protein network. These data point to a close integration of the Crb complex and Par proteins during photoreceptor morphogenesis and underscore the role of Crb as an apical determinant (Muschalik and Knust, 2011, Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors, J Cell Sci. 124:3715-25).
The evolutionarily conserved Crumbs (Crb) complex is crucial for photoreceptor morphogenesis and homeostasis. Loss of Crb results in light-dependent retinal degeneration, which is prevented by feeding mutant flies carotenoid-deficient medium. This suggests a defect in rhodopsin 1 (Rh1) processing, transport, and/or signaling, causing degeneration; however, the molecular mechanism of this remained elusive. In this paper, we show that myosin V (MyoV) coimmunoprecipitated with the Crb complex and that loss of crb led to severe reduction in MyoV levels, which could be rescued by proteasomal inhibition. Loss of MyoV in crb mutant photoreceptors was accompanied by defective transport of the MyoV cargo Rh1 to the light-sensing organelle, the rhabdomere. This resulted in an age-dependent accumulation of Rh1 in the photoreceptor cell (PRC) body, a well-documented trigger of degeneration. We conclude that Crb protects against degeneration by interacting with and stabilising MyoV, thereby ensuring correct Rh1 trafficking. Our data provide, for the first time, a molecular mechanism for the light-dependent degeneration of PRCs observed in crb mutant retinas (Pocha et al., 2011, Crumbs regulates rhodopsin transport by interacting with and stabilising myosin V, J Cell Biol. 195:827-38.
The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types (Mishra et al., 2012, Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli, Eur J Cell Biol. 91:706-16).
Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localises at the Golgi apparatus and centrosome / basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilises pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP:Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation (Baron-Gaillard et al., 2011, Hook2 is involved in the morphogenesis of the primary cilium. Mol Biol Cell. 22:4549-62).
Although columnar epithelial cells are known to acquire an elongated shape, the mechanisms involved in this morphological feature have not yet been completely elucidated. Using columnar human intestinal Caco2 cells, it was established here that the levels of drebrin E, an actin-binding protein, increase in the terminal web both in vitro and in vivo during the formation of the apical domain. Drebrin E depletion was found to impair cell compaction and elongation processes in the monolayer without affecting cell polarity or the formation of tight junctions. Decreasing the drebrin E levels disrupted the normal subapical F-actin-myosin-IIB- βII-spectrin network and the apical accumulation of EB3, a microtubule-plus-end-binding protein. Decreasing the EB3 levels resulted in a similar elongation phenotype to that resulting from depletion of drebrin E, without affecting cell compaction processes or the pattern of distribution of F-actin-myosin-IIB. In addition, EB3, myosin IIB and βII spectrin were found to form a drebrin-E-dependent complex. Taken together, these data suggest that this complex connects the F-actin and microtubule networks apically during epithelial cell morphogenesis, while drebrin E also contributes to stabilising the actin-based terminal web (Bazellieres et al., 2012, Apico-basal elongation requires a drebrin-E-EB3 complex in columnar human epithelial cells, J Cell Sci. 125:919-31).
(2) Foreground obtained from work package 2, studying the primary defects leading to loss of Muller glia cell photoreceptor interaction in conditional Crb2 knockout retinas and embryos:
In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analyzed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganisation was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalisation was detected throughout the CRB2 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors, and disruption of adherens junctions between photoreceptors, Muller glia and progenitor cells. The number of late born progenitor cells, rod photoreceptors and Muller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late born retinal progenitor cells (Alves et al., 2012, Loss of CRB2 in the mouse retina mimics human Retinitis Pigmentosa due to mutations in the CRB1 gene, Hum Mol Genet. doi: 10.1093/hmg/dds398).
Mutations in the Crumbs homologue 1 (Crb1) gene are associated with Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP12). Mice lacking CRB1 demonstrate mild retinal degeneration. However, mice lacking both CRB1 and CRB2 display severe retinal disorganisation (only 2 nuclear and 1 plexiform layers) and thickening. These pathological features are similar to CRB1 LCA patient retinas. In order to understand why Crb1-Crb2 double knockout retinas are so disorganised, we analyzed the phenotype during retinal development. Crb1 knockout mice were crossed with a retinal Crb2-Chx10Cre knockout mouse strain. Animals of different ages were analyzed. Morphological analysis was done with toluidine blue staining of plastic-embedded sections and electron microscopy. To study possible ectopic localisations and up-/down-regulations of protein expressions, immunohistochemistry (IHC) was done on retinas as well as Western blotting. In adult retinas from mice lacking both CRB1 and CRB2, only the number of the latest born cells (rods, bipolar cells, Muller glia and the latest subtypes of amacrine cells) is increased and equivalently spread through the two nuclear layers. As early as E13.5 double knockout retinas show already gaps in the OLM, accompanied with ectopic localisation of progenitor and postmitotic cells. Furthermore, mislocalisation of Chx10-positive progenitor nuclei increases throughout retinal development. From E15.5 increasing amounts of ectopic phospho-H3 positive cells are found through the entire thickness of the developing retina whereas Crumbs complex and adherens junction members disappeared progressively from the OLM during development. Islet1-positive precursor cells, for amacrine and ganglion cells, remain slightly affected whereas Otx2-positive precursor cells, for photoreceptors, are more ectopically localised and in higher proportion than control retinas at E17.5 corroborating our findings in adults. Total number of apoptotic cells is also increased during development as well as in adult double KO retinas. Loss of both CRB1 and CRB2 results in a more severe phenotype than in the Crb1 and Crb2 knockout mice, suggesting that CRB1 and CRB2 have overlapping functions in retina development. Removing CRB1 and CRB2 proteins induces an increased aberrant proliferation of progenitor cells during development leading to a disorganised and thicker retina in adult (Pellissier et al., 2012, Crb1 and Crb2 Controls Cell Division during Retina Development, ARVO2012, poster 6456/A382).
To assess similarities and differences between the human retinal degeneration caused by mutations in the CRB1 gene and mouse retinopathies caused by the lack of one or more Crumbs complex proteins Crb1 and Crb2. Patients with CRB1-associated retinal degeneration (CRB1-RD) were evaluated clinically and with en face and cross-sectional imaging. Crb1-Crb2 double knock-out mice ('DKOs') were generated by cross-breeding Crb1 knock-out mice with a Crb2-Chx10Cre conditional knock-out line. Crb2-Crb1 DKOs were examined at one, three, and six months of age with confocal scanning-laser ophthalmoscopy (cSLO) to assess retinal morphology and vasculature, spectral-domain optical coherence tomography (SD-OCT) to determine the retinal layer structure, and Ganzfeld electroretinography (ERG) to measure retinal function. Patients with CRB1-RD showed early onset retina-wide severe photoreceptor dysfunction by ERG and retinal disorganisation and thickening by OCT (Jacobson et al., HMG 2003;12:1073 and Aleman et al. IOVS 2011;52:6898). We found that Crb1 knock-out mice crossed with a Crb2-Chx10Cre knock-out line might have a similar retinal phenotype, whereas Crb1 knock-outs alone do not.
It turned out that the fundus in native SLO imaging had initially a regular appearance and only changed when signs of degeneration were present, particularly visible in autofluorescence mode. In contrast, the corresponding SD-OCT analysis revealed from early on that retinal lamination appeared to be limited to a 'generic' inner and outer layer with hardly discernible sublayers. Histological workup suggests extensive cellular dislocation as a possible cause for this lack of organisation. With increasing age, a progressive retinal degeneration became visible. At this point, cSLO imaging revealed an increasing number of lesions that gave the fundus a spotty and patchy appearance, as well as several vascular alterations. In OCT, a thinning of the disorganised retina with age was observed. In line with these results, the functional analysis showed initially reduced, but recordable ERG responses at one month of age, and no discernible responses at later time points. The impairment of the Crumbs complex in human CRB1 deficiency may lead to a unique phenotype that demonstrates an abnormally thick retina and a lack of proper lamination. Here, we show that Crb2-Crb1 DKO mice have a similarly disorganised retina, accompanied by a progressive reduction of the ERG response that is also typical for the human patients. Our results suggest that these mice are a valuable model for CRB1-RD and may help to increase our understanding of the underlying disease mechanisms (Garcia-Garrido et al., 2012, Comparison Between CRB1-Associated Retinal Degeneration in Human Patients and Corresponding Mouse Models, ARVO2012, poster 4574/D1061).
(3) Foreground obtained from work package 3, studying the primary defects leading to loss of Muller glia cell photoreceptor interaction in conditional Pals1 gene silenced (knockdown) retinas and embryos:
The membrane-associated palmitoylated protein 5 (MPP5 or PALS1) is thought to organise intracellular PALS1-CRB-MUPP1 protein scaffolds in the retina that are involved in maintenance of photoreceptor-Muller glia cell adhesion. In humans, the Crumbs homolog 1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, especially PALS1, we generated and analyzed conditional knockdown mice for Pals1. Small irregularly shaped spots were detected throughout the PALS1 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. The electroretinography a- and b-wave was severely attenuated in the aged mutant retinas, suggesting progressive degeneration of photoreceptors. The histological analysis showed abnormal retinal pigment epithelium structure, ectopic photoreceptor nuclei in the subretinal space, an irregular outer limiting membrane, half rosettes of photoreceptors in the outer plexiform layer, and a thinner photoreceptor synaptic layer suggesting improper photoreceptor cell layering during retinal development. The PALS1 deficient retinas showed reduced levels of Crumbs complex proteins adjacent to adherens junctions, upregulation of glial fibrillary acidic protein indicative of gliosis, and persisting programmed cell death after retinal maturation. The phenotype suggests important functions of PALS1 in the retinal pigment epithelium in addition to the neural retina (Park et al., 2011, PALS1 is essential for retinal pigment epithelium structure and neural retina stratification, J Neurosci. 31:17230-41).
Cortical development depends upon tightly controlled cell fate and cell survival decisions that generate a functional neuronal population, but the coordination of these two processes is poorly understood. Here we show that conditional removal of a key apical complex protein, Pals1, causes premature withdrawal from the cell cycle, inducing excessive generation of early-born postmitotic neurons followed by surprisingly massive and rapid cell death, leading to the abrogation of virtually the entire cortical structure. Pals1 loss shows exquisite dosage sensitivity, so that heterozygote mutants show an intermediate phenotype on cell fate and cell death. Loss of Pals1 blocks essential cell survival signals, including the mammalian target of rapamycin (mTOR) pathway, while mTORC1 activation partially rescues Pals1 deficiency. These data highlight unexpected roles of the apical complex protein Pals1 in cell survival through interactions with mTOR signalling (Kim et al., 2010, The apical complex couples cell fate and cell survival to cerebral cortical development, Neuron. 66:69-84).
Membrane Palmitoylated Protein 3 (MPP3) is a member of the Membrane Associated Guanylate Kinase (MAGUK) family and is expressed in retina and brain. In the retina, MPP3 is localised at the subapical region adjacent to the outer limiting membrane (OLM) and in the outer plexiform layer. MPP3 and CRB1 interact directly with PALS1/MPP5 and may form a protein complex or mutual exclusive complexes since MPP3 does not bind CRB1. The aim of this study is to further unravel the role of MPP3 in the retina. We have generated Mpp3 conditional knockout (cKO) mice. We have crossed Mpp3 cKO mice with Rx-Cre mice, to specifically abolish Mpp3 in the retina. To investigate the effect of deletion of MPP3 on retinal morphology and localisation of other proteins in the retina, we performed immunohistochemical, morphological and EM analyses on these retinas. Additionally, we have performed functional analyses using OCT, SLO and ERG. We have exposed six months old Mpp3 cKO mice to moderate levels of light to investigate whether this induced a more severe retinal degeneration. In retinas from Mpp3F/F RxCreTg/+ mice up to six months of age we detected a mild degeneration phenotype: At foci ectopic photoreceptor nuclei were detected in the subretinal space, suggesting that the integrity of the OLM might be compromised at these areas and occasionally rosettes were found. Exposure of six months old Mpp3F/F RxCreTg/+ mice to moderate levels (3000 lux) of white light clearly increased the number of degenerative spots in the retina.
Immunohistochemical analysis revealed that levels of PALS1 at the OLM seemed to be decreased in Mpp3F/F RxCreTg/+ mice compared to control. Additionally, at areas where the integrity of the OLM was compromised we observed a loss of Nectin1 and other adherens junction markers. These data suggest that MPP3 is dispensable for proper morphological development of the retina, but might be required for stabilising PALS1. Additionally, we hypothesise that MPP3, like CRB1, is required for the maintenance of adherens junctions during light exposure. These data suggest that Mpp3 might be a novel candidate gene for inherited retinopathies (Dudok et al., 2012, Mpp3 is Required for Maintenance of Adherens Junctions in the Retina during Light Exposure, ARVO2012, poster 6449/A375).
(4) Foreground obtained from WP 4, development of Muller glia progenitor cell transplantation:
Muller glia cells in fish and birds are potential progenitor cells for retinal neurons, but mammalian Muller glia cells are limited in generating neurons in situ. We have studied the capacity of highly purified Muller glia cells to form, upon retinal transplantation, retinal neurons. Mouse Muller glia cells were dissociated, purified by fluorescence-activated cell sorting (FACS) by use of cell surface antigens and vital dye cell trackers, and transplanted subretinally or intravitreously into the mouse retina. Immunohistochemistry was performed on retinal sections and whole-mount preparations. Immunocytochemical analysis of sorted Muller glia cells showed that these cells express markers for Muller glia cells immediately after sort. Upon subretinal or intravitreal transplantation of unmanipulated cells in Crb1-/- or wild-type recipients, these cells efficiently migrate towards the inner face of the inner nuclear layer (INL) and to the ganglion cell layer (GCL), within nine days. The transplanted Muller glia cells change cell fate; they re-differentiate into choline-acetyl transferase (ChAT) positive starburst amacrine cells (SAC). Whole mount staining and mosaics analysis indicated that the projection and mosaics pattern of the transplanted cells is similar to that of resident SACs. The transplanted cells project to the sub-layer s1/2 and s4/5 in the inner plexiform layer (IPL). Furthermore, we showed that transplantation into wild type mice also led to the re-differentiation of these cells into SACs and efficient migration into the retina. In addition, we have initiated experiments to culture these cells enabling their manipulation by applying factors to guide their differentiation behavior, in order to obtain other retinal cell types upon transplantation. Mammalian Muller glia cells can be purified by FACS using cell surface antigens. Upon retinal transplantation, the mouse Muller glia cells efficiently re-differentiate into retinal neurons. Experiments to repeat the results with human Muller glia cells are ongoing (Wijnholds et al., 2012, Adult Muller Glia Cells Are Efficient Progenitor Cells For Starburst Amacrine Cells, ARVO2012, poster 3972/D836).
(5) Foreground obtained from work package 5, optimisation of AAV6 capsids for specific transduction of Muller glia cells, and optimisation of AAV2/6 hCRB1 clinical gene therapy vector production:
Mutations in the CRB1 gene cause retinal degeneration leading to blindness observed in retinitis pigmentosa and Leber's congenital amaurosis. CRB1 protein is expressed in Muller glial cells of the retina. It localises at the subapical region (SAR) just above the adherens junctions between photoreceptor cells and Muller cells. Human CRB1 gene therapy involves
i) the specific targeting of Muller glial cells (specific capsids and/or promoters),
ii) fit in 5Kb of the packaging limitation size of AAV and
iii) correct localisation of CRB1 protein at the SAR and rescue of Crb1-/- mouse models. No expression of hCRB1 protein was found in Muller glial cells transduced with AAV6-CMV-hCRB1 (5.2Kb) while mRNA expression is at similar levels than GFP. AAV6-derived capsids, ShH10-Y445F transduce mainly and efficiently Muller glial cells. 3kb of the Muller and RPE cell specific promoter, RLBP1 or CRALBP, expressing GFP were synthesised and tested in vivo via intravitreal and subretinal routes. Whereas intravitreal pattern is exclusively restricted to Muller cells, the subretinal pattern shows transduction of mainly RPE and Muller cells. CRB1In5: This smaller hCRB1 codon optimised protein is deleted of some EGF domains and contains 56bp splicing donor and acceptor of intron 5. Upon subretinal injection with AAV2/5, CRB1 protein is detected in Crb1-/- retinas in photoreceptor and Muller cells and show a severe phenotype in half of the retina (probably due to over-expression of CRB1 in RPE cells). CRB1 protein is also detected upon intravitreal injection in Muller cells. CRB1coIn5 expression upon AAV injection is correctly localised and spliced in mouse retina. This vector is used and compared to GFP vectors to rescue Crb1-/- and Crb1-/- Crb2F/+ Chx10Cre/+ mice, dKO versus control mice and in toxicity experiment in WT C57Bl/6 mice. CRB1 protein upon AAV injection in murine eye is at detectable level and is correctly spliced and localised. Rescue experiments are ongoing (Pellissier et al., 2012, Human CRB1 gene therapy, CRUMBS IN SIGHT meeting Marseille).
The human CRB1 gene contains 12 exons. 66% of mutations arise in exon 7. Therefore, exon 7 and the first half of exon 9. For cost efficient mutation analysis of the CRB1 gene it is therefore attractive to sequence exons 7 to 9. We sequenced a population of 45 cases of LCA and found 15.5% cases due to mutations in CRB1. We sequenced a population of 710 cases of RP and found 3.5% cases due to mutations in CRB1 (Cremers et al., 2012, presentation at ERDC meeting).
Potential impact:
Community added value and contribution to EU policies. Retinitis pigmentosa comprises a clinically and genetically heterogeneous group of eye diseases that afflicts approximately 1.5 million people worldwide. Affected individuals experience night blindness from early childhood and suffer from progressive photoreceptor degeneration. Macular involvement occurs as the disease progresses and therefore patients experience severe visual impairment in the second decade of life. Patients with Leber congenital amaurosis even suffer from blindness at birth or shortly thereafter. It is estimated that CRB1-associated disease occurs in approximately 3% of patients with RP and in approximately 11% of patients with LCA, affecting more than approximately 3,000 individuals in Europe. CRB2 and genes encoding CRB-interacting-proteins such as PALS1 might also contribute to CRB associated retinal dystrophy. The incidence of CRB1 associated RP12 and LCA in different populations in Europe (e.g. in the province North-Holland, The Netherlands; regions in Spain) indicate that this is a European-wide problem, that results in high costs to society because of loss of labor, and necessary alterations required to the patients' environment to help improve their quality of life. Unfortunately, as of yet, there is no cure available to solve this dramatic situation, and because of increased mobility in Europe, European solutions (molecular diagnostics, cures) are necessary to limit the negative impact of the disease.
We need to pool the geographically spread expertise that is present and available across Europe. In addition to being beneficial to patients, such integration will have other significant advantages. These include an increase in knowledge and competitiveness in Europe and the development of cures that will be used in Europe. It will provide high quality jobs and education possibilities for European scientists, and will attract excellent non-European scientists. It is evident that well trained European scientists are necessary to underpin and set up healthy product pipelines, small-medium enterprises as well as strengthen the European pharmaceutical industry. A high competitor position of the European pharmaceutical industry is important for the technology transfer and the careers of well-trained junior scientists to prevent leakage of knowledge and personnel resources to non-European countries. Clearly, our research fulfils the EU policies to ensure a high level of protection and improvement of consumers' health and safety as well as safeguarding and promoting economic interests.
The six different groups involved in the CRUMBS IN SIGHT project each bring in their specific expertise but have also adapted their research programs to prevent overlap of research, to, distribute the work in a practical, efficient and well-balanced way. The expected impact of carrying out the work at the European level is significantly greater than the sum of the impact of the individual national projects since pre-publication information and materials have been shared. For example, knowledge on the type of proteins interacting with Drosophila Crb, will lead to the identification of proteins interacting with human CRB1, which will lead to an earlier understanding of the mechanism of function of CRB1, and facilitate an earlier development of therapeutics against RP and LCA. It is obvious that this pre-publication information cannot be obtained at the national level since the complementary research facilities and groups with complementary fields of (high quality) expertise do not exist at this level. Therefore, the whole community benefits from our well-balanced consortium as each participant will perform tasks for which they are most suited.
The European added value of the consortium also resides in the establishment of a critical mass in human and financial terms (e.g. all the participants have both the people with specific knowledge and expertise as well as the resource infrastructure appropriate for executing their specialities). Furthermore, the added value resides in the combination of complementary expertise and resources. The participants complemented their work on CRB1 to reduce the impact of retinitis pigmentosa on individuals and populations by fostering the integration of basic and ophthalmological research. All necessary resources for doing the complementary work are available only in the organisations that have the experts.
Our project has been executed by a European consortium, producing knowledge and materials for the European market, and it creates jobs for personnel in the consortium. Additional jobs will be created once we transfer our academic knowledge and materials to the European industry. In fact, technology transfer is likely: Small-medium enterprises are candidates for further Research and Technological Development on our results. Patents will be applied for by the consortium, so technology is protected and exploited in Europe. This transfer of technology to industry improves the European employment but has also impact on improved economic growth compared to non-European countries where modern biotechnology is also already well implemented in industry (e.g. the US and Japan).
Contribution to community social objectives
Loss of vision affects almost everybody at some stage of their life and always results in loss of quality of life regardless of age. The potential results of the proposed study have immediate biological significance, and we anticipate in the long run:
a) Molecular diagnostics for the early detection of the eye disease. Newly identified genes may be specific for or associated with a particular eye disorder.
b) Improvement of quality of life for affected individuals. They will be able to live a more 'normal' life and maintain interactions with other members of the society.
c) Increased security of affected people with regard to job status after gene therapy. The continuation or prolongation of employment for patients with eye disorders after successful treatment has a direct effect on the employment status of these patients.
d) Improved psychological well-being for large groups of patients and (voluntary) organisations that support methods to fight blindness. This is because they are aware that scientists are actively working on understanding and identifying cures for treating visual defects.
e) The knowledge and materials that the consortium will be exploited by technology transfer to a candidate small-medium enterprise that will perform further research and technological development based on our results. This will lead to increased job possibilities and high level technological training. The consortium itself will also profit from the project since our multidisciplinary consortium will obtain extra expertise and insight, and their personnel will have additional training at a technological as well as intellectual level (that is not normally available to basic scientists). The latter again improves the competitiveness and possibilities of the personnel of the consortium in the European job market. All participants will use personal career development plans from the start of the project, and to be updated yearly, to track the career development of the researchers employed on the EC project.
Blindness severely lowers the quality of life of both young and old people. The project contributes to improving quality of life since novel therapeutics directed against the onset of progressive RP and LCA aims to prevent or postpone the loss of vision that occurs in progressive RP patients in the second decade of life and in LCA patients at birth. It should also improve the quality of health of those individuals with a genetic predisposition to other forms of RP, since therapeutics directed against progressive RP/LCA caused by mutations in the CRB1 gene may prevent or delay the onset of disease in these people.
The project contributes to improving the safety of people with RP/LCA since the prevention of the onset of the disease will enable these individuals to function normally in the society (e.g. travelling by car to socialise, handling potentially dangerous equipment in both the work place and at home (e.g. for cooking), providing equal chances on the employment market). In addition, the therapeutics will help to give the RP patients the possibilities for prolonged employment in jobs where basic visual capabilities are necessary.
Therapeutics (viral gene transfer vectors or transplantable Muller glia progenitor cells) shown to be safe and effective in mice to prevent the onset of RP may be an interesting starting point to set up a small company in a European country or to strengthen the current work of a SME. The project will give the opportunity to the groups directly involved in the consortium, plus other individuals in the scientific institutes were the projects are being performed to become educated and trained in state of the art techniques that will have a major impact on the development of the scientific fields of molecular biology, genetics and biomedicine, e.g. the development of gene therapy vectors and Muller glia progenitor cell therapy to treat biochemical diseases.
Plan for the use and dissemination of foreground
The coordinating participant (Dr Wijnholds) is a permanent staff member of the Netherlands Institute for Neuroscience (NIN). The objective of the institute is to perform multidisciplinary fundamental and strategic research concerning the visual system and the brain. It is the objective of the CRUMBS IN SIGHT project consortium to identify therapeutics against diseases of the eye, to stimulate dissemination of knowledge about the cause and possible treatment of eye diseases, and to stimulate the exploitation of results or materials that may cure eye diseases. Therefore, we previously linked up to Amsterdam Molecular Therapeutics B.V. (AMT) as a partner in the CRUMBS IN SIGHT consortium and as a candidate for technology transfer. AMT however left the consortium and recently reorganised into uniQure but remains a serious candidate for further development of the technology. Development of a clinical AAV hCRB1 gene therapy vector will be executed in collaboration with uniQure and/or the facility of Prof. Robin Ali (UCL).
UniQure is a biotechnology company, based in Amsterdam, the Netherlands, which focuses on the development and production of viral products. UniQure has the experience and capabilities to scale the production of different kind of viruses (e.g. adeno-associated virus) to the extent needed to treat patients in future clinical trials. Furthermore uniQure has the experience and capabilities to execute preclinical and clinical development programmes and to guide and execute patenting and licensing strategies. If the current project leads to a suitable AAV hCRB1 gene therapy vector to prevent the onset of eye diseases, uniQure will collaborate to explore whether these genes can become part of a gene therapeutic strategy to cure eye diseases.
Founded in 1998 on the initiative of researchers from the Academic Medical Center (AMC) in Amsterdam, uniQure has become a leader in the field of human gene therapy. A pioneer in the development of gene therapy, uniQure has used its scientific know-how and expertise to build up a strong research and clinical development program filling an unmet medical need for therapy of severe (orphan) diseases in the following areas: metabolic disorders, ocular diseases, central and peripheral nervous system. UniQure is located on the premises of the Academic Medical Center (AMC) in Amsterdam and employs a staff of over 35 highly educated people with scientific and industrial experience. UniQure has state-of-the-art GMP facilities as well as R&D and QC laboratories. UniQure possesses expert capabilities and knowledge covering viral vector generation, scale-up of production processes and manufacturing of clinical grade products. The infrastructure at uniQure is available to companies and academic institutes for GMP manufacturing services of viral products on a fee-for-service basis.
The consortium, driven by the mission of the NIN and scientific and technological challenges, will stimulate the exploitation of the results by a small-medium enterprise such as uniQure on a contract or partnership basis. If financially feasible (after fund raising from e.g. fighting-blindness-organisations, pharmaceutical industry, national governmental departments of economic affairs; or other funds stimulating the set-up of new small-enterprises), we will contract uniQure for the development and production of the cures. In such a scenario, the consortium will control the patents and licenses itself and set up an alternative small enterprise at one of the science parks that are underpinned and supported by the national governments in order to create marketing opportunities. Otherwise, we will ask a small-medium enterprise such as uniQure for a shared partnership. The decision how the consortium will proceed will be made as soon as the final AAV hCRB1 gene therapy vector is determined. Then a detailed technology implementation plan will be set up. In principle, the consortium is willing to transfer the patented knowledge and materials to a European small, medium or large enterprise, such as uniQure, if that is the most beneficial manner to support the well-being of the patients and our efforts in scientific and technological development.
The consortium will also involve uniQure and/or the facilities of Prof. Robin Ali (UCL) to further develop the candidate therapeutics in a demonstration project. The results will be disseminated in patents to improve competitiveness. We published our results in high impact scientific journals, as well as through a web site. The user groups for publication are other scientists at scientific enterprises that would like to obtain a license, as well as universities and research institutes that want to use our materials and knowledge to improve science and technological development for general purposes. We will protect our interests with standard Material and Transfer Agreements.
The Royal Netherlands Academy of Arts and Sciences, to which the NIN belongs, has filed patents in the past and stimulates scientists to file for patents. The licensing of patents to pharmaceutical companies may provide the consortium eventually with additional financial resources to do more research and may provide the community with useful therapeutics. The consortium will join equally in the profits from licensing the patents to pharmaceutical companies or other (not non-profit) institutions.
Knowledge that may significantly improve the technology for usage of the therapeutics or that may significantly improve the therapeutics itself, shall be used to file secondary patents to protect the first patent. Licenses for technology will only be given to serious pharmaceutical companies. As described above the partners agree to extend their co-operation in the exploitation phase by increasing their knowledge on CRB1 and the identification of novel candidate (viral gene) therapeutics against progressive RP and LCA.
The NIN combined with the Academic Medical Center has the infrastructure (e.g. research human ophthalmologists and other medical practicioners, ophthalmological equipment, patients) to initiate and perform future clinical trials in collaboration (or 'pay per batch') with a small-medium enterprise specialised in gene therapy, such as uniQure, that works under biosafe and controlled conditions. Alternatively or at the same time, we will use the infrastructure of Prof. Robin Ali (UCL) to initiate and perform future clinical trials at the Institute of Ophthalmology in London.
The current project linked up with a Dutch high quality demonstration project, a ZonMw gene therapy program by Dr. Jan Wijnholds at the NIN that started 2006, with the goal for demonstration of proof of principle for AAV hCRB1 gene therapy using mice lacking CRB proteins. The CRUMBS IN SIGHT RTD project contributed significantly to the ongoing gene therapy program.
Not all the mechanistic aspects of a functional protein have been elucidated within the four year timescale. However, we do expect that our work will lead to cures. Therefore, after the period financed by the EC and if financially feasible, the consortium as a whole will continue to exploit the results to gain more knowledge on:
a) the pathogenic mechanisms due to loss of interactions between Muller glia cell and photoreceptors leading to RP and LCA,
b) the mechanism of action of all CRB1-interacting proteins since these may represent novel RP disease genes,
c) the elucidation of the function of signalling domains (e.g. the epidermal growth factor domains) in CRB1, and
d) novel candidate proteins to be used as a therapeutic treatment,
e) developing improved methods for viral gene therapy or Muller glia progenitor stem cell therapy (e.g. biosafety in production and application, specificity and duration of expression, application methods).
These follow-on projects of the consortium may be in the form of a new research and technological development project or a demonstration project.
Project website: http://crfb.univ-mrs.fr/Crumbs
Retinitis pigmentosa (RP) denotes a clinically and genetically heterogeneous group of hereditary retinal degenerations. Typical RP begins already early in life with night blindness and a progressive loss of peripheral vision due to successive degeneration of rod photoreceptors. The subsequent degeneration of cone photoreceptors at more progressed stages of RP (which often are reached at quite young ages) results in the loss of useful central vision and blindness. Although approximately 1 in 3000 people is affected by RP and allied disorders, no drugs or therapeutics against these diseases are currently available.
The Crumbs homolog 1 (CRB1) gene is mutated in progressive types of RP, in Leber congenital amaurosis (LCA) and in RP with Coat's-like exsudative vasculopathy. In progressive RP due to autosomal recessive mutations in the CRB1 gene, patients experience night blindness from early childhood and a progressive loss of visual field starting in the second decade of life. The quality of life of LCA patients is even more affected, since they are generally born blind or lose their sight shortly after birth. The CRB1 protein shows high homology to the Crumbs protein previously identified in the fruit-fly Drosophila melanogaster. The Crumbs trans-membrane protein acts as a key player in epithelial polarisation and cell-cell adhesion, prevents degeneration of photoreceptors. Similarly, in human and mouse retinas, CRB1 is required to prevent loss of adhesion between Muller glia cells and photoreceptors, pointing to essential interactions between these two cell types for survival of photoreceptor cells. CRB1 is localised specifically in Muller glia cells. The CRB2 and CRB3 proteins are likely to have partially redundant functions in the retina with CRB1.
The consortium used convenient neurobiological model systems (Muller glia cells and photoreceptors; fly photoreceptor cells) that enabled us to gain insight into neuron-glia interactions in general. We determined the cellular effects of over-expression, mis-expression, loss-of-expression and silencing of other CRB-interacting proteins (MPP5/PALS1, MPP3, MUPP1 and PATJ) in primary retina cultures, polarised cell lines, and Drosophila tissues using endogenous cell polarity markers and functional assays. We investigated the redundancy in CRB1 and CRB2 function, and MPP5 and MPP3 function, in Muller glia H photoreceptor interactions, the precise time of onset of the disease, and the cellular and molecular changes occurring during the onset of the disease. We explored the use of Muller glia progenitor cells for retinal transplantation, and setup adeno-associated virus (AAV)-based gene therapy for the transduction of Muller glia cells, and optimised the production of AAV hCRB1 gene therapy vectors for future clinical use.
We conclude that fruit fly Crumbs prevents photoreceptor cell degeneration by stabilising an unconventional Myosin, MyoV, and hence contributes to proper Rhodopsin transport. In cultured cells, there is a post-translational balance between PALS1, PATJ and MUPP1. Mouse CRB2, MPP5/PALS1 and MPP3 play together with CRB1 important roles in neuron-glia interactions in the retina. Mouse CRB1 and CRB2 regulate the suppression of proliferation of late born retinal progenitors, and loss of both CRB1 and CRB2 result in overgrowth of retina tissue. Muller glia progenitor cells have potential use for retinal transplantation. We developed AAV hCRB1 gene therapy vectors to strengthen Muller glia cell photoreceptor interactions, and which will be further developed for clinical use.
Project Context and Objectives:
Retinitis pigmentosa (RP) denotes a clinically and genetically heterogeneous group of hereditary retinal degenerations. Typical RP begins already early in life with night blindness and a progressive loss of peripheral vision due to successive degeneration of rod photoreceptors. The subsequent degeneration of cone photoreceptors at more progressed stages of RP (which often are reached at quite young ages) results in the loss of useful central vision and blindness. Although approximately 1 in 3000 people is affected by RP and allied disorders, no drugs or therapeutics against these diseases are currently available.
The Crumbs homolog 1 (CRB1) gene is mutated in progressive types of RP, in Leber congenital amaurosis (LCA) and in RP with Coat's-like exsudative vasculopathy. In progressive RP due to autosomal recessive mutations in the CRB1 gene, patients experience night blindness from early childhood and a progressive loss of visual field starting in the second decade of life. The quality of life of LCA patients is even more affected, since they are generally born blind or lose their sight shortly after birth. Immunohistochemical studies on retinas revealed that the mouse and human CRB1 protein is exclusively localised at so-called subapical regions (SARs) adjacent to adherens junctions (AJs) between Muller glia (MG) cells and photoreceptors (PRs) at the outer limiting membrane (OLM). Immuno-electron microscopy on mouse retinas furthermore revealed that CRB1 protein is exclusively present at the SAR of Muller glia cells, whereas CRB family members CRB2 and CRB3 and CRB-interacting proteins localise at the SAR of Muller glia cells and photoreceptors.
The CRB1 protein shows high homology to the Crumbs protein previously identified in the fruitfly Drosophila melanogaster. The Crumbs transmembrane protein acts as a key player in epithelial polarisation and cell-cell adhesion, and prevents degeneration of photoreceptors. Similarly, in human and mouse retinas, CRB1 is required to prevent loss of adhesion between Muller glia cells and photoreceptors, pointing to essential interactions between these two cell types for survival of photoreceptor cells. The CRB2 and CRB3 proteins are likely to have partially redundant functions in the retina with CRB1.
The consortium uses a convenient neurobiological model system (Muller glia cells and photoreceptors; fly photoreceptor cells) that enables us to gain insight into neuron-glia interactions in general. We have integrated high quality expertise that is available across Europe in order to unravel the biochemical and pathophysiological pathways that lead to retinal degeneration in progressive RP and LCA. With the knowledge obtained in this project it will be possible to devise and optimise novel therapies, which will already commence within the timeframe of this project.
It is the overall aim of the CRUMBS IN SIGHT consortium to decipher the function of CRB1 in Muller glia cell photoreceptor interactions, and to develop therapies to restore Muller glia cell photoreceptor interactions mediated by CRB1 in the visual system. The EC proposal can be divided into the following five key objectives addressed in five Work Packages (WP1-WP5):
WP1) Determination of the cellular function of Drosophila, mouse, and human CRB and CRB interacting-proteins (CIPs) in polarised cells and prevention of photoreceptor death.
WP2) Studying the primary defects leading to loss of Muller glia cell photoreceptor interaction in conditional Crb2 knockout retinas and embryos.
WP3) Studying the primary defects leading to loss of Muller glia cell photoreceptor interaction in conditional Pals1 gene silenced (knockdown) retinas and embryos.
WP4) Development of Muller glia progenitor cell transplantation.
WP5) Optimisation of AAV6 capsids for specific transduction of Muller glia cells, and optimisation of AAV2/6 hCRB1 clinical gene therapy vector production.
The proposed experiments are complementary to an ongoing viral hCRB1 gene therapy programme at the Netherlands Institute for Neuroscience. Our expectations are the following: We expect to obtain insight in the function of the CRB complex in regulation of Muller glia cell photoreceptor interaction. We expect to develop safe and efficient therapeutic strategies for cures for neurosensory eye disorders. Our approaches are the following:
WP1 will determine the cellular function of Drosophila, mouse, and human CRB and CRB interacting-proteins (CIPs) in polarised cells and prevention of photoreceptor death. Recent evidence indicated that the CRB-interacting protein PALS1 (MPP5; Stardust) is required for correct localisation of CRB, CRB1, CRB2, and CRB3 as well as several other CRB-interacting proteins at the SAR in mammalian retina and the stalk membrane in adult Drosophila photoreceptors. PALS1 is a member of the membrane associated guanylate kinase- (MAGUK) family of scaffolding proteins. The cellular effects of over-expression, mis-expression and silencing of other CRB-interacting proteins will be evaluated in primary retina cultures, polarised cell lines, and Drosophila tissues using endogenous cell polarity markers and functional assays.
WP2 will analyze retinas and embryos lacking a functional Crb2 gene. Recently we successfully generated and analyzed two Crb1 mutants, and showed an important function for CRB1 in Muller glia cell photoreceptor interactions. CRB1 colocalises with CRB2 and CRB3 at the SAR of Muller glia cells. As recent evidence indicated an important regulatory role for CRB proteins in the formation of tight junctions adjacent to adherens junctions in polarised epithelial cell lines, we will generate conditional Crb2 knockout mice. The mouse model shall help us to investigate redundancy in CRB function in Muller glia cell photoreceptor interactions, the precise time of onset of the disease, and the cellular and molecular changes occurring during the onset of the disease. Crb1/Crb2 double knockout mice will be used to test the efficacy of viral CRB1 gene therapy vectors generated in WP5.
WP3 focuses on the analysis of retinas and embryos lacking a functional Pals1 gene. Previously, we showed by Pals1 gene silencing that PALS1 is required for correct localisation of CRB1, CRB2, CRB3, MUPP1 and VELI3 at the SAR adjacent to adherens junctions at the outer limiting membrane in primary cultured retinas. However, cells that lacked PALS1 still showed morphologically normal Muller glia cell photoreceptor interactions. As it is conceivable that a correct localisation of the CRB1 complex at the SAR is needed for regular functionality, we will generate conditional Pals1 silenced mice. The mouse model shall help us to understand the function of the CRB complex in Muller glia cell photoreceptor interactions, and the cellular and molecular changes occurring during the onset of the disease.
WP4 will study establishment and maintenance of Muller glia cell photoreceptor interaction after subretinal injection of Muller glia cell progenitor cells, and will develop Muller glia progenitor cell therapy. Recently, one of our collaborators showed efficient photoreceptor progenitor transplantation and increased synaptic activity in the mouse eye. A photoreceptor transplantation strategy will not work in patients with mutations in the CRB1 gene because the origin of the defect resides in the Muller glia cells. We are capable of purifying Muller glia cells expressing GFP from different retinal stages, and inject them into the subretinal space. We will determine the optimal differentiation stage of Muller glia cells to be used for transplantation, and study the interaction of the GFP-tagged Muller glia cell with the photoreceptors. In rescue studies, we will investigate the efficacy of this approach to prevent or delay the onset of retinal degeneration in Crb1 mutant mice, particularly with regard to a potential future application in patients with the corresponding neurosensory eye disease.
WP5 will setup adeno-associated virus (AAV)-based gene therapy for the transduction of Muller glia cells, and will optimise the production of clinical AAV2/6 hCRB1 gene therapy vectors. Recently, we identified an AAV serotype capable of efficiently transducing Muller glia cells when intravitreally injected. We will use molecular evolution techniques on the AAV6 capsid to generate AAV serotypes capable of specifically transducing Muller glia cells. Recently, we developed a baculovirus production platform for the AAV1 serotype that is now used for production of clinical (GMP) grade vector. We will develop a similar platform for the AAV6 serotype. We expect to develop therapies (Muller glia stem cell transplantation and AAV CRB1 gene therapy) against progressive RP and LCA that may be used or further developed for clinical trials.
The CRUMBS IN SIGHT project is of vital importance to generate novel therapies against retinal degeneration. The knowledge obtained about the Muller glia cell photoreceptor interaction, and the generated materials and protocols in Muller glia cell transplantation and AAV-based CRB1 gene therapy will be patented, published and disseminated by the consortium. The results will be exploited by technology transfer and by starting a demonstration project in collaboration with a small-medium enterprise.
Project results:
(1) Foreground obtained from work package 1, the determination of the cellular function of Drosophila, mouse, and human CRB and CRB interacting-proteins (CIPs) in polarised cells and prevention of photoreceptor death:
Morphogenesis of Drosophila photoreceptor cells includes the subdivision of the apical membrane into the photosensitive rhabdomere and the associated stalk membrane, as well as a considerable elongation of the cell. Drosophila Crumbs (Crb), an evolutionarily conserved transmembrane protein, organises an apical protein scaffold, which is required for elongation of the photoreceptor cell and extension of the stalk membrane. To further elucidate the role played by different Crb domains during eye morphogenesis, we performed a structure-function analysis in the eye. The analysis showed that the three variants tested, namely full-length Crb, the membrane-bound intracellular domain and the extracellular domain were able to rescue the elongation defects of crb mutant rhabdomeres. However, only full-length Crb and the membrane-bound intracellular domain could partially restore the length of the stalk membrane, while the extracellular domain failed to do so. This failure was associated with the inability of the extracellular domain to recruit beta(Heavy)-spectrin to the stalk membrane. These results highlight the functional importance of the extracellular domain of Crb in the Drosophila eye. They are in line with previous observations, which showed that mutations in the extracellular domain of human CRB1 are associated with retinitis pigmentosa 12 and Leber congenital amaurosis, two severe forms of retinal dystrophy (Richard et al., 2009, A role for the extracellular domain of Crumbs in morphogenesis of Drosophila photoreceptor cells, Eur J Cell Biol. 88:765-77).
Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organise supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organisation is ideally suited to organise protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localised at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis (Bulgakova et al., 2010, Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells, Mol Biol Cell. 21:3915-25).
The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localisation. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localisation of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localisation. We also show a novel function for retromer in maintaining epithelial cell polarity (Pocha et al., 2011, Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs, Curr Biol. 21:1111-7).
Photoreceptor morphogenesis in Drosophila requires remodeling of apico-basal polarity and adherens junctions (AJs), and includes cell shape changes, as well as differentiation and expansion of the apical membrane. The evolutionarily conserved transmembrane protein Crumbs (Crb) organises an apical membrane-associated protein complex that controls photoreceptor morphogenesis. Expression of the small cytoplasmic domain of Crb in crb mutant photoreceptor cells (PRCs) rescues the crb mutant phenotype to the same extent as the full-length protein. Here, we show that over-expression of the membrane-tethered cytoplasmic domain of Crb in otherwise wild-type photoreceptor cells has major effects on polarity and morphogenesis. Whereas early expression causes severe abnormalities in apico-basal polarity and ommatidial integrity, expression at later stages affects the shape and positioning of AJs. This result supports the importance of Crb for junctional remodeling during morphogenetic changes. The most pronounced phenotype observed upon early expression is the formation of ectopic apical membrane domains, which often develop into a complete second apical pole, including ectopic AJs. Induction of this phenotype requires members of the Par protein network. These data point to a close integration of the Crb complex and Par proteins during photoreceptor morphogenesis and underscore the role of Crb as an apical determinant (Muschalik and Knust, 2011, Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors, J Cell Sci. 124:3715-25).
The evolutionarily conserved Crumbs (Crb) complex is crucial for photoreceptor morphogenesis and homeostasis. Loss of Crb results in light-dependent retinal degeneration, which is prevented by feeding mutant flies carotenoid-deficient medium. This suggests a defect in rhodopsin 1 (Rh1) processing, transport, and/or signaling, causing degeneration; however, the molecular mechanism of this remained elusive. In this paper, we show that myosin V (MyoV) coimmunoprecipitated with the Crb complex and that loss of crb led to severe reduction in MyoV levels, which could be rescued by proteasomal inhibition. Loss of MyoV in crb mutant photoreceptors was accompanied by defective transport of the MyoV cargo Rh1 to the light-sensing organelle, the rhabdomere. This resulted in an age-dependent accumulation of Rh1 in the photoreceptor cell (PRC) body, a well-documented trigger of degeneration. We conclude that Crb protects against degeneration by interacting with and stabilising MyoV, thereby ensuring correct Rh1 trafficking. Our data provide, for the first time, a molecular mechanism for the light-dependent degeneration of PRCs observed in crb mutant retinas (Pocha et al., 2011, Crumbs regulates rhodopsin transport by interacting with and stabilising myosin V, J Cell Biol. 195:827-38.
The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types (Mishra et al., 2012, Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli, Eur J Cell Biol. 91:706-16).
Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localises at the Golgi apparatus and centrosome / basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilises pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP:Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation (Baron-Gaillard et al., 2011, Hook2 is involved in the morphogenesis of the primary cilium. Mol Biol Cell. 22:4549-62).
Although columnar epithelial cells are known to acquire an elongated shape, the mechanisms involved in this morphological feature have not yet been completely elucidated. Using columnar human intestinal Caco2 cells, it was established here that the levels of drebrin E, an actin-binding protein, increase in the terminal web both in vitro and in vivo during the formation of the apical domain. Drebrin E depletion was found to impair cell compaction and elongation processes in the monolayer without affecting cell polarity or the formation of tight junctions. Decreasing the drebrin E levels disrupted the normal subapical F-actin-myosin-IIB- βII-spectrin network and the apical accumulation of EB3, a microtubule-plus-end-binding protein. Decreasing the EB3 levels resulted in a similar elongation phenotype to that resulting from depletion of drebrin E, without affecting cell compaction processes or the pattern of distribution of F-actin-myosin-IIB. In addition, EB3, myosin IIB and βII spectrin were found to form a drebrin-E-dependent complex. Taken together, these data suggest that this complex connects the F-actin and microtubule networks apically during epithelial cell morphogenesis, while drebrin E also contributes to stabilising the actin-based terminal web (Bazellieres et al., 2012, Apico-basal elongation requires a drebrin-E-EB3 complex in columnar human epithelial cells, J Cell Sci. 125:919-31).
(2) Foreground obtained from work package 2, studying the primary defects leading to loss of Muller glia cell photoreceptor interaction in conditional Crb2 knockout retinas and embryos:
In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analyzed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganisation was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalisation was detected throughout the CRB2 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors, and disruption of adherens junctions between photoreceptors, Muller glia and progenitor cells. The number of late born progenitor cells, rod photoreceptors and Muller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late born retinal progenitor cells (Alves et al., 2012, Loss of CRB2 in the mouse retina mimics human Retinitis Pigmentosa due to mutations in the CRB1 gene, Hum Mol Genet. doi: 10.1093/hmg/dds398).
Mutations in the Crumbs homologue 1 (Crb1) gene are associated with Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP12). Mice lacking CRB1 demonstrate mild retinal degeneration. However, mice lacking both CRB1 and CRB2 display severe retinal disorganisation (only 2 nuclear and 1 plexiform layers) and thickening. These pathological features are similar to CRB1 LCA patient retinas. In order to understand why Crb1-Crb2 double knockout retinas are so disorganised, we analyzed the phenotype during retinal development. Crb1 knockout mice were crossed with a retinal Crb2-Chx10Cre knockout mouse strain. Animals of different ages were analyzed. Morphological analysis was done with toluidine blue staining of plastic-embedded sections and electron microscopy. To study possible ectopic localisations and up-/down-regulations of protein expressions, immunohistochemistry (IHC) was done on retinas as well as Western blotting. In adult retinas from mice lacking both CRB1 and CRB2, only the number of the latest born cells (rods, bipolar cells, Muller glia and the latest subtypes of amacrine cells) is increased and equivalently spread through the two nuclear layers. As early as E13.5 double knockout retinas show already gaps in the OLM, accompanied with ectopic localisation of progenitor and postmitotic cells. Furthermore, mislocalisation of Chx10-positive progenitor nuclei increases throughout retinal development. From E15.5 increasing amounts of ectopic phospho-H3 positive cells are found through the entire thickness of the developing retina whereas Crumbs complex and adherens junction members disappeared progressively from the OLM during development. Islet1-positive precursor cells, for amacrine and ganglion cells, remain slightly affected whereas Otx2-positive precursor cells, for photoreceptors, are more ectopically localised and in higher proportion than control retinas at E17.5 corroborating our findings in adults. Total number of apoptotic cells is also increased during development as well as in adult double KO retinas. Loss of both CRB1 and CRB2 results in a more severe phenotype than in the Crb1 and Crb2 knockout mice, suggesting that CRB1 and CRB2 have overlapping functions in retina development. Removing CRB1 and CRB2 proteins induces an increased aberrant proliferation of progenitor cells during development leading to a disorganised and thicker retina in adult (Pellissier et al., 2012, Crb1 and Crb2 Controls Cell Division during Retina Development, ARVO2012, poster 6456/A382).
To assess similarities and differences between the human retinal degeneration caused by mutations in the CRB1 gene and mouse retinopathies caused by the lack of one or more Crumbs complex proteins Crb1 and Crb2. Patients with CRB1-associated retinal degeneration (CRB1-RD) were evaluated clinically and with en face and cross-sectional imaging. Crb1-Crb2 double knock-out mice ('DKOs') were generated by cross-breeding Crb1 knock-out mice with a Crb2-Chx10Cre conditional knock-out line. Crb2-Crb1 DKOs were examined at one, three, and six months of age with confocal scanning-laser ophthalmoscopy (cSLO) to assess retinal morphology and vasculature, spectral-domain optical coherence tomography (SD-OCT) to determine the retinal layer structure, and Ganzfeld electroretinography (ERG) to measure retinal function. Patients with CRB1-RD showed early onset retina-wide severe photoreceptor dysfunction by ERG and retinal disorganisation and thickening by OCT (Jacobson et al., HMG 2003;12:1073 and Aleman et al. IOVS 2011;52:6898). We found that Crb1 knock-out mice crossed with a Crb2-Chx10Cre knock-out line might have a similar retinal phenotype, whereas Crb1 knock-outs alone do not.
It turned out that the fundus in native SLO imaging had initially a regular appearance and only changed when signs of degeneration were present, particularly visible in autofluorescence mode. In contrast, the corresponding SD-OCT analysis revealed from early on that retinal lamination appeared to be limited to a 'generic' inner and outer layer with hardly discernible sublayers. Histological workup suggests extensive cellular dislocation as a possible cause for this lack of organisation. With increasing age, a progressive retinal degeneration became visible. At this point, cSLO imaging revealed an increasing number of lesions that gave the fundus a spotty and patchy appearance, as well as several vascular alterations. In OCT, a thinning of the disorganised retina with age was observed. In line with these results, the functional analysis showed initially reduced, but recordable ERG responses at one month of age, and no discernible responses at later time points. The impairment of the Crumbs complex in human CRB1 deficiency may lead to a unique phenotype that demonstrates an abnormally thick retina and a lack of proper lamination. Here, we show that Crb2-Crb1 DKO mice have a similarly disorganised retina, accompanied by a progressive reduction of the ERG response that is also typical for the human patients. Our results suggest that these mice are a valuable model for CRB1-RD and may help to increase our understanding of the underlying disease mechanisms (Garcia-Garrido et al., 2012, Comparison Between CRB1-Associated Retinal Degeneration in Human Patients and Corresponding Mouse Models, ARVO2012, poster 4574/D1061).
(3) Foreground obtained from work package 3, studying the primary defects leading to loss of Muller glia cell photoreceptor interaction in conditional Pals1 gene silenced (knockdown) retinas and embryos:
The membrane-associated palmitoylated protein 5 (MPP5 or PALS1) is thought to organise intracellular PALS1-CRB-MUPP1 protein scaffolds in the retina that are involved in maintenance of photoreceptor-Muller glia cell adhesion. In humans, the Crumbs homolog 1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, especially PALS1, we generated and analyzed conditional knockdown mice for Pals1. Small irregularly shaped spots were detected throughout the PALS1 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. The electroretinography a- and b-wave was severely attenuated in the aged mutant retinas, suggesting progressive degeneration of photoreceptors. The histological analysis showed abnormal retinal pigment epithelium structure, ectopic photoreceptor nuclei in the subretinal space, an irregular outer limiting membrane, half rosettes of photoreceptors in the outer plexiform layer, and a thinner photoreceptor synaptic layer suggesting improper photoreceptor cell layering during retinal development. The PALS1 deficient retinas showed reduced levels of Crumbs complex proteins adjacent to adherens junctions, upregulation of glial fibrillary acidic protein indicative of gliosis, and persisting programmed cell death after retinal maturation. The phenotype suggests important functions of PALS1 in the retinal pigment epithelium in addition to the neural retina (Park et al., 2011, PALS1 is essential for retinal pigment epithelium structure and neural retina stratification, J Neurosci. 31:17230-41).
Cortical development depends upon tightly controlled cell fate and cell survival decisions that generate a functional neuronal population, but the coordination of these two processes is poorly understood. Here we show that conditional removal of a key apical complex protein, Pals1, causes premature withdrawal from the cell cycle, inducing excessive generation of early-born postmitotic neurons followed by surprisingly massive and rapid cell death, leading to the abrogation of virtually the entire cortical structure. Pals1 loss shows exquisite dosage sensitivity, so that heterozygote mutants show an intermediate phenotype on cell fate and cell death. Loss of Pals1 blocks essential cell survival signals, including the mammalian target of rapamycin (mTOR) pathway, while mTORC1 activation partially rescues Pals1 deficiency. These data highlight unexpected roles of the apical complex protein Pals1 in cell survival through interactions with mTOR signalling (Kim et al., 2010, The apical complex couples cell fate and cell survival to cerebral cortical development, Neuron. 66:69-84).
Membrane Palmitoylated Protein 3 (MPP3) is a member of the Membrane Associated Guanylate Kinase (MAGUK) family and is expressed in retina and brain. In the retina, MPP3 is localised at the subapical region adjacent to the outer limiting membrane (OLM) and in the outer plexiform layer. MPP3 and CRB1 interact directly with PALS1/MPP5 and may form a protein complex or mutual exclusive complexes since MPP3 does not bind CRB1. The aim of this study is to further unravel the role of MPP3 in the retina. We have generated Mpp3 conditional knockout (cKO) mice. We have crossed Mpp3 cKO mice with Rx-Cre mice, to specifically abolish Mpp3 in the retina. To investigate the effect of deletion of MPP3 on retinal morphology and localisation of other proteins in the retina, we performed immunohistochemical, morphological and EM analyses on these retinas. Additionally, we have performed functional analyses using OCT, SLO and ERG. We have exposed six months old Mpp3 cKO mice to moderate levels of light to investigate whether this induced a more severe retinal degeneration. In retinas from Mpp3F/F RxCreTg/+ mice up to six months of age we detected a mild degeneration phenotype: At foci ectopic photoreceptor nuclei were detected in the subretinal space, suggesting that the integrity of the OLM might be compromised at these areas and occasionally rosettes were found. Exposure of six months old Mpp3F/F RxCreTg/+ mice to moderate levels (3000 lux) of white light clearly increased the number of degenerative spots in the retina.
Immunohistochemical analysis revealed that levels of PALS1 at the OLM seemed to be decreased in Mpp3F/F RxCreTg/+ mice compared to control. Additionally, at areas where the integrity of the OLM was compromised we observed a loss of Nectin1 and other adherens junction markers. These data suggest that MPP3 is dispensable for proper morphological development of the retina, but might be required for stabilising PALS1. Additionally, we hypothesise that MPP3, like CRB1, is required for the maintenance of adherens junctions during light exposure. These data suggest that Mpp3 might be a novel candidate gene for inherited retinopathies (Dudok et al., 2012, Mpp3 is Required for Maintenance of Adherens Junctions in the Retina during Light Exposure, ARVO2012, poster 6449/A375).
(4) Foreground obtained from WP 4, development of Muller glia progenitor cell transplantation:
Muller glia cells in fish and birds are potential progenitor cells for retinal neurons, but mammalian Muller glia cells are limited in generating neurons in situ. We have studied the capacity of highly purified Muller glia cells to form, upon retinal transplantation, retinal neurons. Mouse Muller glia cells were dissociated, purified by fluorescence-activated cell sorting (FACS) by use of cell surface antigens and vital dye cell trackers, and transplanted subretinally or intravitreously into the mouse retina. Immunohistochemistry was performed on retinal sections and whole-mount preparations. Immunocytochemical analysis of sorted Muller glia cells showed that these cells express markers for Muller glia cells immediately after sort. Upon subretinal or intravitreal transplantation of unmanipulated cells in Crb1-/- or wild-type recipients, these cells efficiently migrate towards the inner face of the inner nuclear layer (INL) and to the ganglion cell layer (GCL), within nine days. The transplanted Muller glia cells change cell fate; they re-differentiate into choline-acetyl transferase (ChAT) positive starburst amacrine cells (SAC). Whole mount staining and mosaics analysis indicated that the projection and mosaics pattern of the transplanted cells is similar to that of resident SACs. The transplanted cells project to the sub-layer s1/2 and s4/5 in the inner plexiform layer (IPL). Furthermore, we showed that transplantation into wild type mice also led to the re-differentiation of these cells into SACs and efficient migration into the retina. In addition, we have initiated experiments to culture these cells enabling their manipulation by applying factors to guide their differentiation behavior, in order to obtain other retinal cell types upon transplantation. Mammalian Muller glia cells can be purified by FACS using cell surface antigens. Upon retinal transplantation, the mouse Muller glia cells efficiently re-differentiate into retinal neurons. Experiments to repeat the results with human Muller glia cells are ongoing (Wijnholds et al., 2012, Adult Muller Glia Cells Are Efficient Progenitor Cells For Starburst Amacrine Cells, ARVO2012, poster 3972/D836).
(5) Foreground obtained from work package 5, optimisation of AAV6 capsids for specific transduction of Muller glia cells, and optimisation of AAV2/6 hCRB1 clinical gene therapy vector production:
Mutations in the CRB1 gene cause retinal degeneration leading to blindness observed in retinitis pigmentosa and Leber's congenital amaurosis. CRB1 protein is expressed in Muller glial cells of the retina. It localises at the subapical region (SAR) just above the adherens junctions between photoreceptor cells and Muller cells. Human CRB1 gene therapy involves
i) the specific targeting of Muller glial cells (specific capsids and/or promoters),
ii) fit in 5Kb of the packaging limitation size of AAV and
iii) correct localisation of CRB1 protein at the SAR and rescue of Crb1-/- mouse models. No expression of hCRB1 protein was found in Muller glial cells transduced with AAV6-CMV-hCRB1 (5.2Kb) while mRNA expression is at similar levels than GFP. AAV6-derived capsids, ShH10-Y445F transduce mainly and efficiently Muller glial cells. 3kb of the Muller and RPE cell specific promoter, RLBP1 or CRALBP, expressing GFP were synthesised and tested in vivo via intravitreal and subretinal routes. Whereas intravitreal pattern is exclusively restricted to Muller cells, the subretinal pattern shows transduction of mainly RPE and Muller cells. CRB1In5: This smaller hCRB1 codon optimised protein is deleted of some EGF domains and contains 56bp splicing donor and acceptor of intron 5. Upon subretinal injection with AAV2/5, CRB1 protein is detected in Crb1-/- retinas in photoreceptor and Muller cells and show a severe phenotype in half of the retina (probably due to over-expression of CRB1 in RPE cells). CRB1 protein is also detected upon intravitreal injection in Muller cells. CRB1coIn5 expression upon AAV injection is correctly localised and spliced in mouse retina. This vector is used and compared to GFP vectors to rescue Crb1-/- and Crb1-/- Crb2F/+ Chx10Cre/+ mice, dKO versus control mice and in toxicity experiment in WT C57Bl/6 mice. CRB1 protein upon AAV injection in murine eye is at detectable level and is correctly spliced and localised. Rescue experiments are ongoing (Pellissier et al., 2012, Human CRB1 gene therapy, CRUMBS IN SIGHT meeting Marseille).
The human CRB1 gene contains 12 exons. 66% of mutations arise in exon 7. Therefore, exon 7 and the first half of exon 9. For cost efficient mutation analysis of the CRB1 gene it is therefore attractive to sequence exons 7 to 9. We sequenced a population of 45 cases of LCA and found 15.5% cases due to mutations in CRB1. We sequenced a population of 710 cases of RP and found 3.5% cases due to mutations in CRB1 (Cremers et al., 2012, presentation at ERDC meeting).
Potential impact:
Community added value and contribution to EU policies. Retinitis pigmentosa comprises a clinically and genetically heterogeneous group of eye diseases that afflicts approximately 1.5 million people worldwide. Affected individuals experience night blindness from early childhood and suffer from progressive photoreceptor degeneration. Macular involvement occurs as the disease progresses and therefore patients experience severe visual impairment in the second decade of life. Patients with Leber congenital amaurosis even suffer from blindness at birth or shortly thereafter. It is estimated that CRB1-associated disease occurs in approximately 3% of patients with RP and in approximately 11% of patients with LCA, affecting more than approximately 3,000 individuals in Europe. CRB2 and genes encoding CRB-interacting-proteins such as PALS1 might also contribute to CRB associated retinal dystrophy. The incidence of CRB1 associated RP12 and LCA in different populations in Europe (e.g. in the province North-Holland, The Netherlands; regions in Spain) indicate that this is a European-wide problem, that results in high costs to society because of loss of labor, and necessary alterations required to the patients' environment to help improve their quality of life. Unfortunately, as of yet, there is no cure available to solve this dramatic situation, and because of increased mobility in Europe, European solutions (molecular diagnostics, cures) are necessary to limit the negative impact of the disease.
We need to pool the geographically spread expertise that is present and available across Europe. In addition to being beneficial to patients, such integration will have other significant advantages. These include an increase in knowledge and competitiveness in Europe and the development of cures that will be used in Europe. It will provide high quality jobs and education possibilities for European scientists, and will attract excellent non-European scientists. It is evident that well trained European scientists are necessary to underpin and set up healthy product pipelines, small-medium enterprises as well as strengthen the European pharmaceutical industry. A high competitor position of the European pharmaceutical industry is important for the technology transfer and the careers of well-trained junior scientists to prevent leakage of knowledge and personnel resources to non-European countries. Clearly, our research fulfils the EU policies to ensure a high level of protection and improvement of consumers' health and safety as well as safeguarding and promoting economic interests.
The six different groups involved in the CRUMBS IN SIGHT project each bring in their specific expertise but have also adapted their research programs to prevent overlap of research, to, distribute the work in a practical, efficient and well-balanced way. The expected impact of carrying out the work at the European level is significantly greater than the sum of the impact of the individual national projects since pre-publication information and materials have been shared. For example, knowledge on the type of proteins interacting with Drosophila Crb, will lead to the identification of proteins interacting with human CRB1, which will lead to an earlier understanding of the mechanism of function of CRB1, and facilitate an earlier development of therapeutics against RP and LCA. It is obvious that this pre-publication information cannot be obtained at the national level since the complementary research facilities and groups with complementary fields of (high quality) expertise do not exist at this level. Therefore, the whole community benefits from our well-balanced consortium as each participant will perform tasks for which they are most suited.
The European added value of the consortium also resides in the establishment of a critical mass in human and financial terms (e.g. all the participants have both the people with specific knowledge and expertise as well as the resource infrastructure appropriate for executing their specialities). Furthermore, the added value resides in the combination of complementary expertise and resources. The participants complemented their work on CRB1 to reduce the impact of retinitis pigmentosa on individuals and populations by fostering the integration of basic and ophthalmological research. All necessary resources for doing the complementary work are available only in the organisations that have the experts.
Our project has been executed by a European consortium, producing knowledge and materials for the European market, and it creates jobs for personnel in the consortium. Additional jobs will be created once we transfer our academic knowledge and materials to the European industry. In fact, technology transfer is likely: Small-medium enterprises are candidates for further Research and Technological Development on our results. Patents will be applied for by the consortium, so technology is protected and exploited in Europe. This transfer of technology to industry improves the European employment but has also impact on improved economic growth compared to non-European countries where modern biotechnology is also already well implemented in industry (e.g. the US and Japan).
Contribution to community social objectives
Loss of vision affects almost everybody at some stage of their life and always results in loss of quality of life regardless of age. The potential results of the proposed study have immediate biological significance, and we anticipate in the long run:
a) Molecular diagnostics for the early detection of the eye disease. Newly identified genes may be specific for or associated with a particular eye disorder.
b) Improvement of quality of life for affected individuals. They will be able to live a more 'normal' life and maintain interactions with other members of the society.
c) Increased security of affected people with regard to job status after gene therapy. The continuation or prolongation of employment for patients with eye disorders after successful treatment has a direct effect on the employment status of these patients.
d) Improved psychological well-being for large groups of patients and (voluntary) organisations that support methods to fight blindness. This is because they are aware that scientists are actively working on understanding and identifying cures for treating visual defects.
e) The knowledge and materials that the consortium will be exploited by technology transfer to a candidate small-medium enterprise that will perform further research and technological development based on our results. This will lead to increased job possibilities and high level technological training. The consortium itself will also profit from the project since our multidisciplinary consortium will obtain extra expertise and insight, and their personnel will have additional training at a technological as well as intellectual level (that is not normally available to basic scientists). The latter again improves the competitiveness and possibilities of the personnel of the consortium in the European job market. All participants will use personal career development plans from the start of the project, and to be updated yearly, to track the career development of the researchers employed on the EC project.
Blindness severely lowers the quality of life of both young and old people. The project contributes to improving quality of life since novel therapeutics directed against the onset of progressive RP and LCA aims to prevent or postpone the loss of vision that occurs in progressive RP patients in the second decade of life and in LCA patients at birth. It should also improve the quality of health of those individuals with a genetic predisposition to other forms of RP, since therapeutics directed against progressive RP/LCA caused by mutations in the CRB1 gene may prevent or delay the onset of disease in these people.
The project contributes to improving the safety of people with RP/LCA since the prevention of the onset of the disease will enable these individuals to function normally in the society (e.g. travelling by car to socialise, handling potentially dangerous equipment in both the work place and at home (e.g. for cooking), providing equal chances on the employment market). In addition, the therapeutics will help to give the RP patients the possibilities for prolonged employment in jobs where basic visual capabilities are necessary.
Therapeutics (viral gene transfer vectors or transplantable Muller glia progenitor cells) shown to be safe and effective in mice to prevent the onset of RP may be an interesting starting point to set up a small company in a European country or to strengthen the current work of a SME. The project will give the opportunity to the groups directly involved in the consortium, plus other individuals in the scientific institutes were the projects are being performed to become educated and trained in state of the art techniques that will have a major impact on the development of the scientific fields of molecular biology, genetics and biomedicine, e.g. the development of gene therapy vectors and Muller glia progenitor cell therapy to treat biochemical diseases.
Plan for the use and dissemination of foreground
The coordinating participant (Dr Wijnholds) is a permanent staff member of the Netherlands Institute for Neuroscience (NIN). The objective of the institute is to perform multidisciplinary fundamental and strategic research concerning the visual system and the brain. It is the objective of the CRUMBS IN SIGHT project consortium to identify therapeutics against diseases of the eye, to stimulate dissemination of knowledge about the cause and possible treatment of eye diseases, and to stimulate the exploitation of results or materials that may cure eye diseases. Therefore, we previously linked up to Amsterdam Molecular Therapeutics B.V. (AMT) as a partner in the CRUMBS IN SIGHT consortium and as a candidate for technology transfer. AMT however left the consortium and recently reorganised into uniQure but remains a serious candidate for further development of the technology. Development of a clinical AAV hCRB1 gene therapy vector will be executed in collaboration with uniQure and/or the facility of Prof. Robin Ali (UCL).
UniQure is a biotechnology company, based in Amsterdam, the Netherlands, which focuses on the development and production of viral products. UniQure has the experience and capabilities to scale the production of different kind of viruses (e.g. adeno-associated virus) to the extent needed to treat patients in future clinical trials. Furthermore uniQure has the experience and capabilities to execute preclinical and clinical development programmes and to guide and execute patenting and licensing strategies. If the current project leads to a suitable AAV hCRB1 gene therapy vector to prevent the onset of eye diseases, uniQure will collaborate to explore whether these genes can become part of a gene therapeutic strategy to cure eye diseases.
Founded in 1998 on the initiative of researchers from the Academic Medical Center (AMC) in Amsterdam, uniQure has become a leader in the field of human gene therapy. A pioneer in the development of gene therapy, uniQure has used its scientific know-how and expertise to build up a strong research and clinical development program filling an unmet medical need for therapy of severe (orphan) diseases in the following areas: metabolic disorders, ocular diseases, central and peripheral nervous system. UniQure is located on the premises of the Academic Medical Center (AMC) in Amsterdam and employs a staff of over 35 highly educated people with scientific and industrial experience. UniQure has state-of-the-art GMP facilities as well as R&D and QC laboratories. UniQure possesses expert capabilities and knowledge covering viral vector generation, scale-up of production processes and manufacturing of clinical grade products. The infrastructure at uniQure is available to companies and academic institutes for GMP manufacturing services of viral products on a fee-for-service basis.
The consortium, driven by the mission of the NIN and scientific and technological challenges, will stimulate the exploitation of the results by a small-medium enterprise such as uniQure on a contract or partnership basis. If financially feasible (after fund raising from e.g. fighting-blindness-organisations, pharmaceutical industry, national governmental departments of economic affairs; or other funds stimulating the set-up of new small-enterprises), we will contract uniQure for the development and production of the cures. In such a scenario, the consortium will control the patents and licenses itself and set up an alternative small enterprise at one of the science parks that are underpinned and supported by the national governments in order to create marketing opportunities. Otherwise, we will ask a small-medium enterprise such as uniQure for a shared partnership. The decision how the consortium will proceed will be made as soon as the final AAV hCRB1 gene therapy vector is determined. Then a detailed technology implementation plan will be set up. In principle, the consortium is willing to transfer the patented knowledge and materials to a European small, medium or large enterprise, such as uniQure, if that is the most beneficial manner to support the well-being of the patients and our efforts in scientific and technological development.
The consortium will also involve uniQure and/or the facilities of Prof. Robin Ali (UCL) to further develop the candidate therapeutics in a demonstration project. The results will be disseminated in patents to improve competitiveness. We published our results in high impact scientific journals, as well as through a web site. The user groups for publication are other scientists at scientific enterprises that would like to obtain a license, as well as universities and research institutes that want to use our materials and knowledge to improve science and technological development for general purposes. We will protect our interests with standard Material and Transfer Agreements.
The Royal Netherlands Academy of Arts and Sciences, to which the NIN belongs, has filed patents in the past and stimulates scientists to file for patents. The licensing of patents to pharmaceutical companies may provide the consortium eventually with additional financial resources to do more research and may provide the community with useful therapeutics. The consortium will join equally in the profits from licensing the patents to pharmaceutical companies or other (not non-profit) institutions.
Knowledge that may significantly improve the technology for usage of the therapeutics or that may significantly improve the therapeutics itself, shall be used to file secondary patents to protect the first patent. Licenses for technology will only be given to serious pharmaceutical companies. As described above the partners agree to extend their co-operation in the exploitation phase by increasing their knowledge on CRB1 and the identification of novel candidate (viral gene) therapeutics against progressive RP and LCA.
The NIN combined with the Academic Medical Center has the infrastructure (e.g. research human ophthalmologists and other medical practicioners, ophthalmological equipment, patients) to initiate and perform future clinical trials in collaboration (or 'pay per batch') with a small-medium enterprise specialised in gene therapy, such as uniQure, that works under biosafe and controlled conditions. Alternatively or at the same time, we will use the infrastructure of Prof. Robin Ali (UCL) to initiate and perform future clinical trials at the Institute of Ophthalmology in London.
The current project linked up with a Dutch high quality demonstration project, a ZonMw gene therapy program by Dr. Jan Wijnholds at the NIN that started 2006, with the goal for demonstration of proof of principle for AAV hCRB1 gene therapy using mice lacking CRB proteins. The CRUMBS IN SIGHT RTD project contributed significantly to the ongoing gene therapy program.
Not all the mechanistic aspects of a functional protein have been elucidated within the four year timescale. However, we do expect that our work will lead to cures. Therefore, after the period financed by the EC and if financially feasible, the consortium as a whole will continue to exploit the results to gain more knowledge on:
a) the pathogenic mechanisms due to loss of interactions between Muller glia cell and photoreceptors leading to RP and LCA,
b) the mechanism of action of all CRB1-interacting proteins since these may represent novel RP disease genes,
c) the elucidation of the function of signalling domains (e.g. the epidermal growth factor domains) in CRB1, and
d) novel candidate proteins to be used as a therapeutic treatment,
e) developing improved methods for viral gene therapy or Muller glia progenitor stem cell therapy (e.g. biosafety in production and application, specificity and duration of expression, application methods).
These follow-on projects of the consortium may be in the form of a new research and technological development project or a demonstration project.
Project website: http://crfb.univ-mrs.fr/Crumbs