Philip M. Zakas

Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction

Optimization of a proteins pharmaceutical properties is usually carried out by rational design and/or directed evolution. Here we test an alternative approach based on ancestral sequence reconstruction. Using available genomic sequence data on coagulation factor VIII and predictive models of molecular evolution, we engineer protein variants with improved activity, stability, and biosynthesis potential and reduced inhibition by anti-drug antibodies. In principle, this approach can be applied to any protein drug based on a conserved gene sequence.

Development and Characterization of Recombinant Ovine Coagulation Factor VIII

Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the worlds population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for the research and development of novel nucleic acid and protein-based therapies for hemophilia A.

Expanding the ortholog approach for hemophilia treatment complicated by factor VIII inhibitors

The formation of neutralizing antibodies (inhibitors) directed against human coagulation factor VIII (hFVIII) is a life‐threatening pathogenic response that occurs in 20–30% of severe congenital hemophilia A patients and 0.00015% of the remaining population (i.e. acquired hemophilia A). Interspecies amino acid sequence disparity among FVIII orthologs represents a promising strategy to mask FVIII from existing inhibitors while retaining procoagulant function. Evidence for the effectiveness of this approach exists in clinical data obtained for porcine FVIII (pFVIII) products, which have demonstrated efficacy in the setting of congenital and acquired hemophilia.

Engineered Hematopoietic Stem Cells as Therapeutics for Hemophilia A.

The field of HSCT gene therapy has advanced from proof-of-concept studies to the treatment of humans with acquired and genetic diseases. Several clinical trials have resulted in life-saving successes, and the curative potential of genetically-modified HSCs is now a reality with the number of disease applications growing rapidly, including chronic granulomatous disease, Wiscott-Aldrich disease, Fanconi anemia, β-thalassemia, and sickle cell disease. Hemophilia A remains a prime candidate for HSCT gene therapy. Advancements in fVIII transgene design, viral vector engineering, and immunological conditioning have cured this disease in animal models and show promise for upcoming clinical trials. Additional studies continue to elucidate methods and technologies to identify and isolate HSCs and improve transduction of this important gene therapy target. In addition, overcoming the current limitations of fVIII expression, as well as reducing the risks of the gene transfer procedure, such as insertional mutagenesis, is possible. Further advancements in the field of HSCT gene therapy have included novel mechanisms to target site-specific gene insertions or corrections using engineered nucleases or PNA-complexes. Trough ongoing intensive research, the field of HSCT gene therapy is progressing towards safer, more efficient, and cost effective treatment options.

Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A

Potency is a key optimization parameter for hemophilia A gene therapy product candidates. Optimization strategies include promoter engineering to increase transcription, codon optimization of mRNA to improve translation, and amino-acid substitution to promote secretion. Herein, we describe both rational and empirical design approaches to the development of a minimally sized, highly potent AAV-fVIII vector that incorporates three unique elements: a liver-directed 146-nt transcription regulatory module, a target-cell-specific codon optimization algorithm, and a high-expression bioengineered fVIII variant. The minimal synthetic promoter allows for the smallest AAV-fVIII vector genome known at 4,832 nt, while the tissue-directed codon optimization strategy facilitates increased fVIII transgene product expression in target cell types, e.g., hepatocytes, over traditional genome-level codon optimization strategies. As a tertiary approach, we incorporated ancient and orthologous fVIII sequence elements previously shown to facilitate improved biosynthesis through post-translational mechanisms. Together, these technologies contribute to an AAV-fVIII vector that confers sustained, curative levels of fVIII at a minimal dose in hemophilia A mice. Moreover, the first two technologies should be generalizable to all liver-directed gene therapy vector designs.

456. Transgene Bioengineering Through Ancestral Protein Reconstruction

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.