Sharon C. Cunningham, Philip M. Zakas, Natsuki Sasaki, Eva B. van Dijk, Erhua Zhu, Yanfang Fu, William E. Salomon, Robert J. Citorik, Jacob R. Rubens, Cecilia Cotta-Ramusino, William Querbes, Ian E. Alexander
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An alternative transposon system, <i>Sleeping Beauty</i>, has been widely used for <i>ex vivo</i> gene delivery; however liver-targeted delivery using a hybrid rAAV/<i>Sleeping Beauty</i> approach remains relatively unexplored.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>We investigated the capacity of a <i>Sleeping Beauty</i> (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC).</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient <i>Spf</i><sup><i>ash</i></sup> mouse following elimination of residual endogenous murine OTC.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>The present study demonstrates the first <i>in vivo</i> utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.</p>\n </section>\n </div>","PeriodicalId":56122,"journal":{"name":"Journal of Gene Medicine","volume":"26 8","pages":""},"PeriodicalIF":3.2000,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system\",\"authors\":\"Sharon C. Cunningham, Philip M. Zakas, Natsuki Sasaki, Eva B. van Dijk, Erhua Zhu, Yanfang Fu, William E. Salomon, Robert J. Citorik, Jacob R. Rubens, Cecilia Cotta-Ramusino, William Querbes, Ian E. Alexander\",\"doi\":\"10.1002/jgm.3726\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Background</h3>\\n \\n <p>Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/<i>piggyBac</i> transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, <i>Sleeping Beauty</i>, has been widely used for <i>ex vivo</i> gene delivery; however liver-targeted delivery using a hybrid rAAV/<i>Sleeping Beauty</i> approach remains relatively unexplored.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods</h3>\\n \\n <p>We investigated the capacity of a <i>Sleeping Beauty</i> (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC).</p>\\n </section>\\n \\n <section>\\n \\n <h3> Results</h3>\\n \\n <p>At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient <i>Spf</i><sup><i>ash</i></sup> mouse following elimination of residual endogenous murine OTC.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Conclusions</h3>\\n \\n <p>The present study demonstrates the first <i>in vivo</i> utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. 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Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system
Background
Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored.
Methods
We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC).
Results
At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC.
Conclusions
The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.
期刊介绍：
The aims and scope of The Journal of Gene Medicine include cutting-edge science of gene transfer and its applications in gene and cell therapy, genome editing with precision nucleases, epigenetic modifications of host genome by small molecules, siRNA, microRNA and other noncoding RNAs as therapeutic gene-modulating agents or targets, biomarkers for precision medicine, and gene-based prognostic/diagnostic studies.
Key areas of interest are the design of novel synthetic and viral vectors, novel therapeutic nucleic acids such as mRNA, modified microRNAs and siRNAs, antagomirs, aptamers, antisense and exon-skipping agents, refined genome editing tools using nucleic acid /protein combinations, physically or biologically targeted delivery and gene modulation, ex vivo or in vivo pharmacological studies including animal models, and human clinical trials.
Papers presenting research into the mechanisms underlying transfer and action of gene medicines, the application of the new technologies for stem cell modification or nucleic acid based vaccines, the identification of new genetic or epigenetic variations as biomarkers to direct precision medicine, and the preclinical/clinical development of gene/expression signatures indicative of diagnosis or predictive of prognosis are also encouraged.
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