Research Models and Gene Augmentation Therapy for CRB1 Retinal Dystrophies

Boon, Nanda; Wijnholds, Jan; Pellissier, Lucie P.

doi:10.3389/fnins.2020.00860

Cited by 17 publications

(18 citation statements)

References 114 publications

(201 reference statements)

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“… 52 We found only neovascularization events in the GCL/NFL with rAAV-h CRB1 that may be linked to differences of h CRB1 over h CRB2 expression in DL-AAA-stressed astrocytes. (3) The protective function of hCRB2 over hCRB1 protein may also be explained by unknown differences in the intracellular protein function (both have short intracellular FERM/PDZ/ERLI domains 9 , 14 ) or differences in hCRB homodimerization/heteromerization of extracellular hCRB/mCRB protein at the OLM. However, both vectors rescued the retinal morphology at the OLM where we would expect that the homodimerization/heteromerization takes place.…”

Section: Discussionmentioning

confidence: 99%

“…hCRB1/mCRB1 (1,406 and 1,405 aa; UniProtKB: P82279 and Q8VHS2, respectively) and hCRB2/mCRB2 (1,285 and 1282 aa; UniProtKB: Q5IJ48 and Q80YA8, respectively) proteins expressed at the OLM are similar in size and have similar domains (signal peptide, EGF-like, LamG, transmembrane, FERM, PDZ ERLI). 9 , 14 The pAAV-h CRB plasmid, pHelper, and pXX2-ShH10F were co-transfected in 10× 15-cm dishes of 80% confluent HEK293T cells to generate rAAV2/ShH10 Y445F .CMV.h CRB2 co.spA or rAAV2/ShH10 Y445F .CMVmin.h CRB1 co.spA viral particles. After benzonase treatment, the lysates were ultracentrifuged onto an iodixanol density gradient.…”

Section: Methodsmentioning

confidence: 99%

“…(1) The knockout (KO) of the Crb1 gene (Crb1 KO ) ablates the expression of the CRB1 protein in MGCs and retinal precursor cells, and it resulted in slow progression of retinal disorganization and degeneration from postnatal day 14 (P14) on. 8,12,14 (2) In Crb1 KO mice, retinal degeneration occurs at foci in the inferior temporal quadrant of the retina. (3) Cell-type specific ablation of CRB1 in MGCs, or of CRB2 in MGCs, suggested that CRB proteins execute important overlapping roles in MGCs.…”

Section: Introductionmentioning

confidence: 99%

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

Buck

Vos

Alves

et al. 2021

Molecular Therapy - Methods & Clinical Development

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Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific Crb1 KO Crb2 LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl -α-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 (h CRB2 ) in Müller cells of Crb1 KO Crb2 LowMGC mice subsequently exposed to low levels of dl -α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (h CRB1 ) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that h CRB expression in Müller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-h CRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-h CRB1 in protection against loss of vision.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Buck

Vos

Alves

et al. 2021

Molecular Therapy - Methods & Clinical Development

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“…In addition, in Crb1 mouse models, the retinal degeneration is limited to the inferior quadrant [3,17], whereas in the Crb1 mutant rats it is presented throughout the entire retina. These discrepancies could be because of (1) different genetic backgrounds, including species differences; (2) different types of mutations affecting different CRB isoforms [21,22], thereby expressing a distinct CRB1 INDEL protein in the Crb1 mutant rats; or (3) the total expression levels of CRB2 might be significantly lower in new-born rats compared to new-born mice. Decreased levels of CRB2 or dysregulation of other CRB-interacting proteins could result in less stabilization of the adherens junction complex at the OLM, resulting in a more severe phenotype.…”

Section: Discussionmentioning

confidence: 99%

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

Boon

Alves

Mulder

et al. 2021

IJMS

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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 Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller 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 ShH10Y445F 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 ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller 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 Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.

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“…The other challenge in CRB1 gene therapy is simultaneous expression of the therapeutic vector in dissimilar cell types that demand CRB function. 42 …”

Section: Exploring Gene Targets In Lcamentioning

confidence: 99%

<p>Voretigene Neparvovec and Gene Therapy for Leber’s Congenital Amaurosis: Review of Evidence to Date</p>

Padhy¹,

Takkar²,

Narayanan³

et al. 2020

TACG

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Gene therapy has now evolved as the upcoming modality for management of many disorders, both inheritable and non-inheritable. Knowledge of genetics pertaining to a disease has therefore become paramount for physicians across most specialities. Inheritable retinal dystrophies (IRDs) are notorious for progressive and relentless vision loss, frequently culminating in complete blindness in both eyes. Leber's congenital amaurosis (LCA) is a typical example of an IRD that manifests very early in childhood. Research in gene therapy has led to the development and approval of voretigene neparvovec (VN) for use in patients of LCA with a deficient biallelic RPE65 gene. The procedure involves delivery of a recombinant virus vector that carries the RPE65 gene in the subretinal space. This comprehensive review reports the evidence thus far in support of gene therapy for LCA. We explore and compare the various gene targets including but not limited to RPE65, and discuss the choice of vector and method for ocular delivery. The review details the evolution of gene therapy with VN in a phased manner, concluding with the challenges that lie ahead for its translation for use in communities that differ much both genetically and economically.

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Research Models and Gene Augmentation Therapy for CRB1 Retinal Dystrophies

Cited by 17 publications

References 114 publications

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

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

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

<p>Voretigene Neparvovec and Gene Therapy for Leber’s Congenital Amaurosis: Review of Evidence to Date</p>

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