In Vivo CRISPR/Cas9-Mediated Genome Editing Mitigates Photoreceptor Degeneration in a Mouse Model of X-Linked Retinitis Pigmentosa

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

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In vivo potency testing of subretinal rAAV5.hCNGB1 gene therapy in the Cngb1 knockout mouse model of retinitis pigmentosa

Human Gene Therapy ◽

10.1089/hum.2021.121 ◽

2021 ◽

Author(s):

Johanna E Wagner ◽

Lena Zobel ◽

Maximilian Joachim Gerhardt ◽

Catherine R O'Riordan ◽

Amy Frederick ◽

...

Keyword(s):

Gene Therapy ◽

Mouse Model ◽

Retinitis Pigmentosa ◽

Knockout Mouse ◽

Knockout Mouse Model

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Dynamic in vivo quantification of rod photoreceptor degeneration using fluorescent reporter mouse models of retinitis pigmentosa

Experimental Eye Research ◽

10.1016/j.exer.2019.107895 ◽

2020 ◽

Vol 190 ◽

pp. 107895 ◽

Cited By ~ 2

Author(s):

Harry O. Orlans ◽

Alun R. Barnard ◽

Robert E. MacLaren

Keyword(s):

Retinitis Pigmentosa ◽

Mouse Models ◽

Rod Photoreceptor ◽

Photoreceptor Degeneration ◽

Fluorescent Reporter ◽

Reporter Mouse

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Robust Factor IX Expression Following ZFN-Mediated Genome Editing in An Adult Mouse Model of Hemophilia B

Blood ◽

10.1182/blood.v118.21.668.668 ◽

2011 ◽

Vol 118 (21) ◽

pp. 668-668

Author(s):

Xavier M Anguela ◽

Rajiv Sharma ◽

Hojun Li ◽

Virginia Haurigot ◽

Anand Bhagwat ◽

...

Keyword(s):

Mouse Model ◽

Genome Editing ◽

Partial Hepatectomy ◽

Transgene Expression ◽

Target Site ◽

Factor Ix ◽

Regulatory Control ◽

Neonatal Mice ◽

Proliferation Of Hepatocytes

Abstract Abstract 668 As a therapeutic strategy, site-specific modification of the genome has the potential to avoid some of the disadvantages of traditional gene replacement approaches such as insertional mutagenesis and lack of endogenous regulatory control of expression. We have recently reported that zinc finger nuclease (ZFN) driven gene correction can be achieved in vivo in a neonatal mouse model of hemophilia by combining AAV-mediated delivery of both the ZFNs and a Factor IX donor template with homology to the targeted F.IX gene (Li et al., Nature, 2011). The mouse model carries a mutant human F.IX mini-gene (hF9mut) knocked into the ROSA26 locus and ZFN-mediated cleavage followed by donor-dependent repair results in restoration of functional F.IX expression. AAV-ZFN and AAV-Donor vectors were administered to neonatal mice, where the rapid proliferation of hepatocytes in the growing animal may promote genome editing through homology directed repair (HDR). Here we sought to investigate whether ZFN-mediated genome editing is feasible in adult animals with predominantly quiescent hepatocytes. Tail vein injection of the AAV-ZFN and AAV-Donor, containing a promoterless wild type factor IX insert flanked by arms of homology to the target site, into adult (8 week old) mice (n=17) resulted in stable (>10wk) circulating F.IX levels of 730–1900 ng/mL (15-38% of normal), whereas mice receiving ZFN alone (n=9) exhibited F.IX levels below detection (<15 ng/mL). Co-delivery of AAV-Mock (luciferase expressing) & AAV-Donor (n=9), yielded <65 ng/mL F.IX. Importantly, mice lacking the hF9mut gene averaged less than 100 ng/mL after receiving AAV-ZFN and AAV-Donor (n=8), suggesting that F.IX expression was derived from on-target genome editing. To eliminate the potential for hF.IX expression resulting from episomal (non-integrated) AAV genomes we performed a two-thirds partial hepatectomy two days after AAV administration. Liver regeneration following hepatectomy is known to substantially reduce expression from non-integrated AAV genomes yet no significant differences in transgene expression were observed compared to non-hepatectomized mice: circulating F.IX levels in the AAV-ZFN + AAV-Donor group (n=13) ranged between 678–1240 ng/mL, whereas mice receiving ZFN alone (n=8) or Mock + AAV-Donor (n=8) had no detectable F.IX expression, or <100 ng/mL F.IX, respectively. Taken together, these data suggest that the F.IX expression in ZFN + Donor treated mice was derived from stable correction of the genome at the intended target site. In summary, we have shown that synchronized cell proliferation of hepatocytes, either in neonatal mice or following partial hepatectomy, is not necessary to achieve highly efficient genome editing and resultant high levels of transgene expression in vivo. These findings substantially expand the potential of ZFN-mediated genome editing as a therapeutic modality. Disclosures: Doyon: Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment.

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Carnosic acid slows photoreceptor degeneration in the Pde6brd10 mouse model of retinitis pigmentosa

Scientific Reports ◽

10.1038/srep22632 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 33

Author(s):

Kai Kang ◽

Matthew J. Tarchick ◽

Xiaoshan Yu ◽

Craig Beight ◽

Ping Bu ◽

...

Keyword(s):

Mouse Model ◽

Retinitis Pigmentosa ◽

Carnosic Acid ◽

Photoreceptor Degeneration

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Retinitis Pigmentosa Results in Neurodegeneration Concomitant With Neuroinflammation, Extracellular Matrix Disorganization and the Upregulation of Neuroprotective Pathways in Glial Cells and Neurons

10.21203/rs.3.rs-101559/v1 ◽

2020 ◽

Author(s):

Christina B. Bielmeier ◽

Saskia Roth ◽

Sabrina I. Schmitt ◽

Stefaniya K. Boneva ◽

Anja Schlecht ◽

...

Keyword(s):

Extracellular Matrix ◽

Retinitis Pigmentosa ◽

Retinal Degeneration ◽

Molecular Mechanisms ◽

Genetic Model ◽

Immunofluorescent Staining ◽

System Response ◽

Photoreceptor Degeneration ◽

The Impact

Abstract BackgroundHereditary retinal degenerations like retinitis pigmentosa (RP) are amongst the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. Here, we in-depth characterized the transgenic VPP mouse model, a genetic model for autosomal dominant RP. MethodsWe examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescent staining and 3D reconstruction. ResultsOne month-old VPP mice showed a significantly higher number of apoptotic photoreceptor cells that resulted in a significantly thinner ONL in three months-old VPP mice, concomitant with an increase in reactivity of microglia and Müller cells. By RNASeq analysis we identified 9,256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, amongst others, dysregulation of TGF-β regulated extracellular matrix organization, factors of the (ocular) immune system/response and apoptosis. ConclusionThe predominant effect pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated and VEGF signaling that were significantly associated with the VPP transgene-induced photoreceptor degeneration. Thus, modulation of these processes might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.

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