Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system.

DNA-based gene therapy has considerable therapeutic potential, but the challenges associated with delivery continue to limit progress. Messenger RNA (mRNA) has the potential to provide for transient production of therapeutic proteins, without the need for nuclear delivery and without the risk of insertional mutagenesis. Here we describe the sustained delivery of therapeutic proteins in vivo in both rodents and non-human primates via nanoparticle-formulated mRNA. Nanoparticles formulated with lipids and lipid-like materials were developed for delivery of two separate mRNA transcripts encoding either human erythropoietin (hEPO) or factor IX (hFIX) protein. Dose-dependent protein production was observed for each mRNA construct. Upon delivery of hEPO mRNA in mice, serum EPO protein levels reached several orders of magnitude (>125 000-fold) over normal physiological values. Further, an increase in hematocrit (Hct) was established, demonstrating that the exogenous mRNA-derived protein maintained normal activity. The capacity of producing EPO in non-human primates via delivery of formulated mRNA was also demonstrated as elevated EPO protein levels were observed over a 72-h time course. Exemplifying the possible broad utility of mRNA drugs, therapeutically relevant amounts of human FIX (hFIX) protein were achieved upon a single intravenous dose of hFIX mRNA-loaded lipid nanoparticles in mice. In addition, therapeutic value was established within a hemophilia B (FIX knockout (KO)) mouse model by demonstrating a marked reduction in Hct loss following injury (incision) to FIX KO mice.

Suitability and utility of computational analysis tools: characterization of erythrocyte parameter variation.

Systems engineering can provide insights into multivariate regulatory networks and pooling in complex biological networks that cannot be fully interpreted through experiments alone. Herein, we analyzed the use of phase planes, modal and time-lagged correlation (TLC) analyses of the human erythrocyte to explore the utility of these techniques for understanding the effect of single parameter changes on the behavior of a metabolic network. Specifically, several parameters in key regulatory steps in erythrocyte glycolysis, Rapoport-Leubering bypass, pentose phosphate pathway, adenosine metabolism, and membrane transport were perturbed. The most sensitive parameters were identified based on the steady-state metabolite concentration changes and were explored further. Modal analysis identified relevant time scales for each parameter change. These time scales were further explored using phase plane and TLC analyses. Phase plane and TLC both inferred pooling changes, while TLC also identified changes in the regulatory network structure that resulted from various parameter changes. Each method has strengths and weaknesses for exploring and gaining insight into complex biological networks.