title: Effects of Self-Complementarity, Codon Optimization, Transgene, and Dose on Liver Transduction with AAV8.
abstract: Numerous methods of vector design and delivery have been employed in an attempt to increase transgene expression following AAV-based gene therapy. Here, a gene transfer study was conducted in mice to compare the effects of vector self-complementarity (double- or single-stranded DNA), codon optimization of the transgene, and vector dose on transgene expression levels in the liver. Two different reporter genes were used: human ornithine transcarbamylase (hOTC) detected by immunofluorescence, and enhanced green fluorescent protein (EGFP) detected by direct fluorescence. The AAV8 capsid was chosen for all experiments due to its strong liver tropism. While EGFP is already a codon-optimized version of the original gene, both wild-type (WT) and codon-optimized (co) versions of the hOTC transgene were compared in this study. In addition, the study evaluated which of the two hOTC modifications-codon optimization or self-complementarity-would confer the highest increase in expression levels at a given dose. Interestingly, based on morphometric image analysis, it was observed that the difference in detectable expression levels between self-complementary (sc) and single-stranded (ss) hOTCco vectors was dose dependent, with a sevenfold increase in OTC-positive area using sc vectors at a dose of 3 × 109genome copies (GC) per mouse, but no significant difference at a dose of 1 × 1010GC/mouse. In contrast, with EGFP as a transgene, the increases in expression levels when using the sc vector were observed at both the 3 × 109GC/mouse and 1 × 1010GC/mouse doses. Furthermore, codon optimization of the hOTC transgene generated a more significant improvement in expression than the use of self-complementarity did. Overall, the results demonstrate that increases in expression levels gained by using sc vectors instead of ss vectors can vary between different transgenes, and that codon optimization of the transgene can have an even more powerful effect on the resulting expression levels.
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: Peter
authors.initials: P
authors.lastname: Bell
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: Shu-Jen
authors.initials: SJ
authors.lastname: Chen
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: Mohamad
authors.initials: M
authors.lastname: Nayal
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: Zhenning
authors.initials: Z
authors.lastname: He
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: John
authors.initials: J
authors.lastname: White
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: Deborah
authors.initials: D
authors.lastname: Lebel-Hagan
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: James M
authors.initials: JM
authors.lastname: Wilson
mesh_terms: D000818:Animals; D003062:Codon; D000229:Dependovirus / Q000235:genetics*; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000627:therapeutic use; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D009954:Ornithine Carbamoyltransferase / Q000235:genetics / Q000627:therapeutic use; D014161:Transduction, Genetic
chemical_list: D003062:Codon; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D009954:Ornithine Carbamoyltransferase
keywords: AAV8; codon optimization; liver gene transfer; self-complementarity