CRB1 gene therapy coming of age: mechanistic insight and rAAV assays on mouse & human retinal organoid models

Biallelic CRB1 gene variations can cause retinitis pigmentosa (RP), Leber congenital amaurosis, or in some cases macular degeneration. This thesis describes the generation and analysis of RP-CRB1 mouse and human retinas (mouse: Crb1KOCrb2LowMGCs; chapter 2. Human RP-CRB1-patient-derived organoids (chapter 4 and 5). The data indicates that the human RP-CRB1 disease can be studied in mice and human organoids. Then, we show that recombinant adeno-associated viral (rAAV)-CRB gene supplementation therapy to Müller glial cells (MGCs) of the Crb1KOCrb2LowMGCs mouse retina can protect it from stress-induced vision loss, and that human CRB2 cDNA was superior to human CRB1 cDNA (chapter 2). We then developed an improved rAAV tropism assay on human donor eyes (chapter 3). This assay shows that rAAV5 can efficiently infect Müller glial cells and photoreceptors, the target cells of a RP-CRB1 gene therapy. Also, rAAV5 infection studies outperformed rAAV9 on human retinal organoids and human donor retinas (chapter 4). Finally, we find much more early endosomes and an increase of the degradative cellular vesicles which is linked to decrease of RAB11A-postive recycling endosomes in RP-CRB1 patient organoids (chapter 5). Thus, this thesis on both human and mouse models provides new insight into retinal degeneration and rAAV gene supplementation therapies. The research here described was conducted at the Department of Ophthalmology, at the Leiden University Medical Center (LUMC). The studies described in this thesis were financially supported by Foundation Fighting Blindness, The Netherlands Organisation for Health Research and Development: ZonMw, Curing Retinal Blindness Foundation, Stichting Retina Nederland Fonds, Landelijke St. Blinden en Slechtzienden, Rotterdamse Stichting Blindenbelangen, St. Blindenhulp, St. Blinden-Penning, Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (ANVVB), Gelderse Blinden Stichting and MaculaFonds.LUMC / Geneeskunde Repositoriu

AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model

Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Muller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Muller cell-specific Crb1(KO)Crb2(LowMGC) retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Muller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of DL-alpha-aminoadipate acid induced gliosis and retinal disorganization in Crb1(KO)Crb2(LowMGC) retinas but not in wild-type or Crb1-deficient retinas. Crb1(KO)Crb2(LowMGC) mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Muller cells of Crb1(KO)Crb2(LowMGC) mice subsequently exposed to low levels of DL-alpha-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Muller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.Ophthalmic researc

Cluster Interpretation of Properties of Alternating Parity Bands in Heavy Nuclei

The properties of the states of the alternating parity bands in actinides, Ba, Ce and Nd isotopes are analyzed within a cluster model. The model is based on the assumption that cluster type shapes are produced by the collective motion of the nuclear system in the mass asymmetry coordinate. The calculated spin dependences of the parity splitting and of the electric multipole transition moments are in agreement with the experimental data.Comment: 29 pages, 10 figure

Experimental Proposal for Achieving Superadditive Communication Capacities with a Binary Quantum Alphabet

We demonstrate superadditivity in the communication capacity of a binary alphabet consisting of two nonorthogonal quantum states. For this scheme, collective decoding is performed two transmissions at a time. This improves upon the previous schemes of Sasaki et al. [Phys. Rev. A 58, 146 (1998)] where superadditivity was not achieved until a decoding of three or more transmissions at a time. This places superadditivity within the regime of a near-term laboratory demonstration. We propose an experimental test based upon an alphabet of low photon-number coherent states where the signal decoding is done with atomic state measurements on a single atom in a high-finesse optical cavity.Comment: 7 pages, 5 figure

Defining phenotype, tropism, and retinal gene therapy using adeno-associated viral vectors (AAVs) in new-born Brown Norway rats with a spontaneous mutation in Crb1

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Muller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Muller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10(Y445F) vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10(Y445F) at P5 or P8 resulted in efficient infection of mainly Muller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10(Y445F) to infect Muller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Muller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.Ophthalmic researc

Primordial nucleosynthesis with a varying fine structure constant: An improved estimate

We compute primordial light-element abundances for cases with fine structure constant alpha different from the present value, including many sources of alpha dependence neglected in previous calculations. Specifically, we consider contributions arising from Coulomb barrier penetration, photon coupling to nuclear currents, and the electromagnetic components of nuclear masses. We find the primordial abundances to depend more weakly on alpha than previously estimated, by up to a factor of 2 in the case of ^7Li. We discuss the constraints on variations in alpha from the individual abundance measurements and the uncertainties affecting these constraints. While the present best measurements of primordial D/H, ^4He/H, and ^7Li/H may be reconciled pairwise by adjusting alpha and the universal baryon density, no value of alpha allows all three to be accommodated simultaneously without consideration of systematic error. The combination of measured abundances with observations of acoustic peaks in the cosmic microwave background favors no change in alpha within the uncertainties.Comment: Phys. Rev. D accepted version; minor changes in response to refere

The Influence of an External Chromomagnetic Field on Color Superconductivity

We study the competition of quark-antiquark and diquark condensates under the influence of an external chromomagnetic field modelling the gluon condensate and in dependence on the chemical potential and temperature. As our results indicate, an external chromomagnetic field might produce remarkable qualitative changes in the picture of the color superconducting (CSC) phase formation. This concerns, in particular, the possibility of a transition to the CSC phase and diquark condensation at finite temperature.Comment: 27 pages, RevTex, 8 figures; the version accepted for the publication in PRD (few references added; new numerical results added; main conclusions are not changed

Random polytopes: Their definition, generation and aggregate properties

The definition of random polytope adopted in this paper restricts consideration to those probability measures satisfying two properties. First, the measure must induce an absolutely continuous distribution over the positions of the bounding hyperplanes of the random polytope; and second, it must result in every point in the space being equally as likely as any other point of lying within the random polytope. An efficient Monte Carlo method for their computer generation is presented together with analytical formulas characterizing their aggregate properties. In particular, it is shown that the expected number of extreme points for such random polytopes increases monotonically in the number of constraints to the limiting case of a polytope topologically equivalent to a hypercube. The implied upper bound of 2 n where n is the dimensionality of the space is significantly less than McMullen's attainable bound on the maximal number of vertices even for a moderate number of constraints.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47911/1/10107_2005_Article_BF01585093.pd

Identification and Functional Characterization of G6PC2 Coding Variants Influencing Glycemic Traits Define an Effector Transcript at the G6PC2-ABCB11 Locus

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights