456. Transgene Bioengineering Through Ancestral Protein Reconstruction
Authors: Philip M. Zakas, Harrison C. Brown, Kristopher A. Knight, Ernest T. Parker, H. Trent Spencer, Eric A. Gaucher, Christopher B. Doering
Journal: Molecular Therapy (2016)

Bioengineering of the transgene often is a critical component of preclinical gene therapy R&D. Transgenes and their products represent the active agent in nucleic acid pharmaceuticals and similar to small molecule pharmaceuticals, they can be modified to possess improved pharmacological properties. However as they are significantly more complex than small molecules, the available strategies for bioengineering, such as in silico rational design, directed evolution and homolog/ortholog-scanning mutagenesis, are less robust. Herein, we propose combined ancestral sequence and protein reconstruction (ASR and APR, respectively) as newly accessible approaches to transgene/transgene product bioengineering. ASR is the prediction of ancient sequences from extant ones and well developed ASR methods and tools now exist. Furthermore, the availability of de novo custom DNA synthesis and recombinant protein expression systems now facilitates APR to complement and extend ASR findings. Previously through the study of extant FVIII orthologs, we discovered that differential molecular, biochemical and immunological properties with exist and could have a positive pharmacological impact upon engineering into human FVIII. For example, porcine FVIII was shown to display 10-100-fold more efficient biosynthesis than human FVIII in vitro and in vivo, while murine FVIII displays 5 - 10-fold greater stability following thrombin activation. Ovine FVIII displays intermediate biosynthesis and stability, but strikingly reduced cross-reactivity to anti-human FVIII inhibitory antibodies. APR provides a high-resolution mapping solution to these ortholog sequence-activity relationships and also takes advantage of the observation that ancient proteins often have unpredicted and/or expanded functionalities that can be efficiently mapped to specific amino acid residues through comparisons of ancestral proteins and genes within an evolutionary lineage. Therefore, we sought to validate APR as a FVIII discovery/bioengineering platform with the expectation that this approach can be successfully applied to essentially all hemostatic, as well as non-hemostatic, gene therapies. Initially, we employed ASR/APR to resurrect 14 ancestral (An) FVIII molecules. Each An-FVIII was shown to be active in standard coagulation assays using human plasma demonstrating evolutionary compatibility. To study biosynthetic efficiency, secreted An-FVIII activity and mRNA transcript levels were analyzed from stably transfected cells demonstrating that, An-53, an ancestral primate sequence with 95% identity to extant human FVIII, displayed the greatest biosynthetic efficiency equivalent to porcine FVIII and our lead bioengineered high expression FVIII, ET3. As a proxy for AAV gene therapy, hemophilia A mice were administered several doses of a liver-directed An-53 AAV plasmid DNA cassette via hydrodynamic injection resulting in peak plasma FVIII activity levels ≥12-fold higher than observed with the ET3 transgene. In addition to superior biosynthetic efficiency, we have identified An-FVIII variants with 2 - 3 fold improved specific activity and stability greater or equal to murine FVIII. Furthermore, we have identified An-FVIII molecules that display reduced immune reactivity and have used these constructs to define functional epitopes to the single amino acid level. Currently, we are refining this approach to identify the key functional residues responsible for each property with the goal of improving the pharmacology of the human FVIII transgenes.