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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

1619: Validating Reliability of Genome Editing Using Ion Channels as Drug Target

Type: Poster Session

Poster Board Number: 1619
Presentation Details
Session Title: Friday Poster Session
Start Time: 5/19/2023 12:00
End Time: 5/19/2023 14:00

Severe forms of inherited ocular channelopathy are caused by point mutations in KCNJ13, which affect the retinal pigment epithelial (RPE). We have previously demonstrated that both gene therapy and translational read-through result in functional recovery as tested using induced pluripotent stem cells iPSC-RPE from a patient line. AAV-gene therapy related immune responses, CRISPR/Cas9 gene editing associated off-targets, and indels pose some challenges in drug discovery and its clinical translation. We used Adenosine and Cytosine base editors (ABE8e, evoCDA-CBE & BE4max-CBE) along with a specific sgRNA as proof-of-concept correction of KCNJ13 point mutations (c.158G>A [p.W53X] and c.431T> C [p.L144P]) using nanoparticle-mediated delivery to HEK293-stable cells, LCA16-patient’s specific fibroblasts and iPSC-RPE. BE efficiency, protein localization and high throughput channel function were assessed in edited cells and compared with non-edited mutant and WT cells. Potential off-targets were screened to evaluate the accuracy and efficacy of BEs. CBE for L144P editing in stable cells resulted in high on-target editing (60-80%). However, bystander 'Cs' in the protospacer region led to either missense (~61%) or silent (~59%) variations in edited cells which affected the gene function. Very few reads had a WT gene sequence (~3%). ABE8e mRNA for W53X correction in stable cells showed 50% editing. Nanoparticle-mediated delivery of ABE8e in fibroblasts (47% editing) and iPSC-RPE (20%) established its use for in vivo BE delivery. On target indel mutagenesis (<3%) and screening of potential off-target sites (<1%) indicated high accuracy of the ABEs. Electrophysiology assays demonstrated in vivo functional rescue in a mouse model. In conclusion, sequence complexity of L144P locus poses some challenges to edit it with the current generation of CRISPR BEs. Using modified CBEs with tighter editing windows or using prime editing for the hard-to-rescue alleles would be beneficial. Functional studies to confirm the therapeutic potential of base editing is must. A caution is warranted to avoid detrimental outcomes of silent editing. W53X editing using ABE8e delivered via non-viral platform, silica nanoparticles showed specific editing without generating detrimental bystander substitutions, indels or off-target edits elsewhere in the genome. K+ conductance in iPSC-RPE confirmed the functional rescue of the Kir7.1 channel following base editing. Altogether, these preclinical base editing studies for correcting mutations causing childhood blindness and other ocular genetic diseases hold promise for clinical translation.

B. Pattnaik1, Meha Kabra2, Pawan Shahi2, David Gamm3, David R. Liu4, Shaoqin S. Gong3, Krishanu Saha5

1Pediatrics, Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI,2Pediatrics,, University of Wisconsin, Madison, WI,3Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI,4Harvard University and HHMI, Cambridge, MA,5Biomedical Engineering, University of Wisconsin, Madison, WI
  B. Pattnaik: 1; Commercial Interest i.e. Company X; Hubble Therapeutics. 1; For what role? i.e. Speaker; Inventor.

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