Sources Of Off-Target Expression From Recombinase-Dependent Aav Vectors And Mitigation With Cross-Over Insensitive Atg-Out Vectors
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA(2019)
摘要
In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or "leak" expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinas-especific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant "cross-over insensitive" recognition sites combined with an "ATG-out" design. We show that these CIAO (crossover insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.
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关键词
AAV, leak, recombinase, Cre, Flp
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