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Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors.

ABSTRACT: 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 recombinase-specific 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 (cross-over 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.

SUBMITTER: Fischer KB

PROVIDER: S-EPMC6936690 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors.

Fischer Kyle B KB Collins Hannah K HK Callaway Edward M EM

Proceedings of the National Academy of Sciences of the United States of America 20191216 52

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 ...[more]

PMID: 31843925

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Project description:BackgroundCOVID-19 mitigation measures intend to protect public health, but their adverse psychological, social, and economic effects weaken public support. Less favorable trade-offs may especially weaken support for more restrictive measures. Support for mitigation measures may also differ between population subgroups who experience different benefits and costs, and decrease over time, a phenomenon termed "pandemic fatigue."MethodsWe examined self-reported support for COVID-19 mitigation measures in the Netherlands over 12 consecutives waves of data collection between April 2020 and May 2021 in an open population cohort study. Participants were recruited through community panels of the 25 regional public health services, and through links to the online surveys advertised on social media. The 54,010 unique participants in the cohort study on average participated in 4 waves of data collection. Most participants were female (65%), middle-aged [57% (40-69 years)], highly educated (57%), not living alone (84%), residing in an urban area (60%), and born in the Netherlands (95%).ResultsCOVID-19 mitigation measures implemented in the Netherlands remained generally well-supported over time [all scores >3 on 5-point scale ranging 1 (low)-5 (high)]. During the whole period studied, support was highest for personal hygiene measures, quarantine and wearing face masks, high but somewhat lower for not shaking hands, testing and self-isolation, and restricting social contacts, and lowest for limiting visitors at home, and not traveling abroad. Women and higher educated people were more supportive of some mitigation measures than men and lower educated people. Older people were more supportive of more restrictive measures than younger people, and support for more socially restrictive measures decreased most over time in higher educated people or in younger people.ConclusionsThis study found no support for pandemic fatigue in terms of a gradual decline in support for all mitigation measures in the first year of the pandemic. Rather, findings suggest that support for mitigation measures reflects a balancing of benefits and cost, which may change over time, and differ between measures and population subgroups.

Project description:Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here, we investigated whether incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3'-untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that five copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and ?-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23-fold in the liver. However, gene expression in other tissues, including the heart was not inhibited. Similarly, we inserted four copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3'-UTR of the AAV-luciferase vector. We wished to see whether they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time, regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly because of promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that liver-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in nonhematopoietic tissues resulted from promoter shut-off.

Dataset Information

Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors.

Publications

Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors.

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