728. Tissue-Directed Transgene Engineering for AAV and Lentivector Gene Therapy Approaches
Authors: Harrison C. Brown, Phillip M. Zakas, Stephan N. George, Ernest T. Parker, H. Trent Spencer, Christopher B. Doering
Journal: Molecular Therapy (2016)

Clinical gene therapy frequently is encumbered by low transgene product biosynthesis at predictably safe vector doses. It has been hypothesized that the presence of rare codons may regulate transgene product expression through depletion of the available cognate tRNA pool. Codon optimization is the prominent strategy utilized to overcome this hypothesized limitation and involves replacing rare, presumably translation rate-limiting, codons with the most frequent ones. Typical algorithms attempt to match the codon usage frequency of the transgene coding sequence to that of target organism's total mRNA pool, which has been shown to approximate the overall available tRNA concentrations. Upon closer examination, it appears that both codon frequency and tRNA content vary between tissue types. Therefore, we hypothesize that codon-optimization can be improved by tailoring to the codon-frequency of the most highly expressed genes present in target cell types. Our tissue-directed codon optimization algorithm utilizes novel codon usage indices generated from target cell gene expression data. As proof of concept, we developed tissue-codon optimized variants of coagulation factor VIII (FVIII) to be utilized in lenti- and adeno-associated viral (LV and AAV, respectively) vectors. These two vectors are the leading platforms for clinical gene therapy of hemophilia A. LV is utilized to target autologous hematopoietic cell types ex vivo while AAV is delivered intravenously to genetically-modify hepatocytes. However to date, development-stage LV and AAV gene therapy products for hemophilia A have been characterized by low-level FVIII transgene product biosynthesis. Initially, we designed human hepatocyte-, monocyte- and standard overall human-optimized FVIII constructs (LCO, MCO and HCO, respectively) to be compared to wild-type FVIII. Upon initial examination, expression of LCO-FVIII was shown to be 3-fold greater than either MCO- or wild-type FVIII from the human hepatoma cell line, HepG2, following transient transfection. In contrast, LCO-FVIII and MCO-FVIII expression was diminished 12 and 4-fold, respectively, compared to wild-type in the 'neutral' human embryonic kidney 293T cell line. Furthermore, following hydrodynamic injection of naked plasmid DNA into hemophilia A mice, LCO-FVIII exhibited a sustained 10-fold increase in FVIII expression relative to the HCO-FVIII comparator. In attempt to generate a lead candidate for clinical translation, we utilized several of our most promising vector components to construct a liver-optimized AAV8 vector consisting of LCO-ET3, our previously described high-expression bioengineered FVIII variant, transcriptionally driven by a novel 146bp liver-directed promoter and adjacent MVM intron. Following intravenous delivery into hemophilia A mice, vector doses of 1e12 and 1e11 vector genomes per kg achieved sustained, predictable curative plasma FVIII levels of 200% and 20% of normal human levels, respectively. These initial results support the utility of our novel approach of clinical tissue-directed transgene optimization.