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8/2/2019 Gene Delivery

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AAV-mediated knockdown

of Peripherin-2 in vivousing miRNA-based

hairpinsA Georgiadis, M Tschernutter, JWB Bainbridge, SJRobbie, J McIntosh, AC Nathwani, AJ Smith and RR Ali

Reporter: Amy Liu

2012. 1.13


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Introduction

Dominantly inherited retinopathy isa subcategory of inherited retinaldisorder

Peripherin-2 (Prph2) is a highlyexpressed photoreceptor- specificgene in which mutations can lead to

dominantly inherited retinal disorder

Gene silencing by RNAi mechanism


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Prph-2

32kDa structural membraneglycoprotein

Localized in the photoreceptor outersegment

It is an essential component for the

rod/ cone outer segment morphology


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Prph2-silencing hairpin were selected from online designwebsite.

Together with non-targeting control (siCON), efficacy wastested in vitro.293T/wtRDS+

HEK cells were infected with a lentiviral vector with Prph2to engineer a 293T-HEK cell line that stably expresses

Prph2

Prph2 is fivefoldhigher than

endogenous -actin293T/wtRDS+

Stable CellLine

In VitroTests


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1. siRDS6 target area and miR-30a template was used todesign a miRNA-based hairpin:miRDS6

2. a conventional shRNA havingsame target area as siRDS6:

shRDS6

miRNA-based design of RNAihairpins has been shown toboth increase efficiency andreduce toxicity.

Silent mutation in the gene can cause evasion of hairpinsilencing.The seed region of RDS6 was altered to abolish shRNA:mRNAbinding, while retaining the wild-type amino acid sequence.

A stably expressing cell linewas constructed by lentiviralinfection with mutated Prph2cDNA: 293T/mutRDS+

In VitroTests


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In VitroTestsmiRDS6, shRDS6 and miRCON hairpin cassettes were cloned

into an AAV2/8 vector backbone, with a red fluorescent protein

(RFP)

plasmid vector

Transfection to 293T/wtRDS+ and 293T/mutRDS+ cell.Sorted by FACS to select RFP+ cell, total RNA was extractedand Prph2 level was determined by qRT-PCR (adjust to -actinlevel)

Prph

2level


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In VivoTestsThree plasmids were used to produce AAV2/8 particle, titer

matched, and then subretinally injected into adult wild type

mice.Three weeks after single injection, retinae was dissociated andRFP+ photoreceptors were sorted by FACS.Total RNA extracted, Prph2 levels were assessed by qRT-PCR (adjustto -actin level)

Prph

2level


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4/20/12 a: single subretinal inj b: AAV2/8.RFP.miRCON c: AAV2/8.RFP.shRDS6 d:

RFP: redfluorescentproteinDAPI: cell nucleusPrph-2: immuno-

staining

Immuno-histologicalAnalysis(3 weeks afterinjection ofPrph-2silencing)


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4/20/12 ONL: outer nuclear layer IS: inner segments OS: outer segments RPE: retinal

Morphological Analysis (5 weeks after injection ofPrph-2 silencing)


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PhotoreceptorQuantification


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Conclusion

The author showed for the first time,a miRNA-based design can efficientlybe used in AAV2/8-mediated

silencing of the photoreceptor genePrph2 in vitro and in vivo.

Whether or not miRNA-based design

is more efficient than conventionallydesigned shRNA is still unanswered.

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