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CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters.

Abstract：

:Validation of gene transfer vectors containing tissue-specific promoters in cell-based functional assays poses a formidable challenge for gene therapy product development. Here, we describe a novel approach based on CRISPR/dCas9 transcriptional activation to achieve robust transgene expression from transgene cassettes containing tissue or cell type-specific promoters after infection with AAV vectors in cell-based systems. Guide RNA sequences targeting two promoters that are highly active within mammalian photoreceptors were screened in a novel promoter activation assay. Using this screen, we generated and characterized stable cell lines that co-express dCas9.VPR and top-performing guide RNA candidates. These cells exhibit potent activation of proviral plasmids after transfection or after infection with AAV vectors delivering transgene cassettes carrying photoreceptor-specific promoters. In addition, we interrogated mechanisms to optimize this platform through the addition of multiple guide RNA sequences and co-expression of the universal adeno-associated virus receptor (AAVR). Collectively, this investigation identifies a rapid and broadly applicable strategy to enhance in vitro expression and to evaluate potency of AAV vectors that rely upon cell or tissue-specific regulatory elements.

journal_name

Mol Ther Methods Clin Dev

journal_title

Molecular therapy. Methods & clinical development

authors

McDougald DS,Duong TT,Palozola KC,Marsh A,Papp TE,Mills JA,Zhou S,Bennett J

doi

10.1016/j.omtm.2019.03.004

subject

Has Abstract

pub_date

2019-03-28 00:00:00

pages

380-389

issn

2329-0501

pii

S2329-0501(19)30031-2

journal_volume

13

pub_type

杂志文章

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