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Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases.

ABSTRACT: Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.

SUBMITTER: De Caneva A

PROVIDER: S-EPMC6693827 | biostudies-other | 2019 Jun

REPOSITORIES: biostudies-other

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Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases.

De Caneva Alessia A Porro Fabiola F Bortolussi Giulia G Sola Riccardo R Lisjak Michela M Barzel Adi A Giacca Mauro M Kay Mark A MA Vlahoviček Kristian K Zentilin Lorena L Muro Andrés F AF

JCI insight 20190618

Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination ...[more]

PMID: 31211694

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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

Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases.

Publications

Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases.

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