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Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.

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Title: Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.
Authors: Andrieu-Soler, C., Halhal, M., Boatright, J.H., Padove, S.A., Nickerson, J.M., Stodulkova, E., Stewart, R.E., Ciavatta, V.T., Doat, M., Jeanny, J.C., de Bizemont, T., Sennlaub, F., Courtois, Y., Behar-Cohen, F.
Publication Year: 2025
Collection: Université de Lausanne (UNIL): Serval - Serveur académique lausannois
Subject Terms: Aging/metabolism, Animals, Newborn, Cyclic Nucleotide Phosphodiesterases, Type 6, Eye/enzymology, Immunohistochemistry/methods, Iontophoresis, Mice, Inbred C3H, Mutant Strains, Oligonucleotides/administration & dosage, Oligonucleotides/therapeutic use, Phosphoric Diester Hydrolases/genetics, Phosphoric Diester Hydrolases/metabolism, Point Mutation, Retina/enzymology, Retina/pathology, Retinal Degeneration/enzymology, Retinal Degeneration/genetics, Rhodopsin/metabolism, Staining and Labeling, Targeted Gene Repair
Description: PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. ...
Document Type: article in journal/newspaper
File Description: application/pdf
Language: English
ISSN: 1090-0535
Relation: Molecular Vision; https://iris.unil.ch/handle/iris/54958; serval:BIB_3568F758A5AE; 000247143900001
Availability: https://iris.unil.ch/handle/iris/54958
Accession Number: edsbas.99F15AFB
Database: BASE
Description
Abstract:PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. ...
ISSN:10900535