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Zone in with Zon—Modified mRNA Mania, Clinical and Commercial Potential

Dr. Gerald Zon’s latest “Zone in with Zon” blog post, dated December 2, 2013, and published by TriLink BioTechnologies of San Diego, provides a fascinating discussion of the developing use of modified mRNAs in a wide variety of key applications, including gene therapy, nucleic acid vaccines, and cellular reprogramming, as well as the possibly tremendous commercial potential of modified mRNA technology in these and other areas. Dr. Zon begins by discussing the intellectual simplicity of gene therapy, i.e., to simply replace a broken gene with the DNA of the normal gene and thus ultimately generate the normal version of the missing or altered protein. Unfortunately, it has proven remarkably difficult over three decades of work to achieve this effectively and safely. Dr. Zon attributes this in part to the challenges for cell- or tissue-specific delivery, as well as concern for adverse events generally ascribed to unintended vector integration leading to neoplasias. Nevertheless, there are presently more than 1700 clinical trials of gene therapy taking place around the world. However, as a consequence of the slow progress of DNA-based gene therapy, a number of researchers have recently turned to the study of modified mRNAs that might be used to produce the missing protein directly by translation. This field is called “mRNA therapeutics.” Another application is in the use of mRNAs as cancer vacccines. Here the idea is to use mRNAs coding for tumor-associated antigens to induce specific immune responses to the tumor. Dr. Zon notes that a 2013 review from Novartis Vaccines & Diagnostics, and entitled “RNA: the New Revolution in Nucleic Acid Vaccines,” claims that the “prospects for success are bright.” The reasons for this, Dr. Zon says, include the potential of RNA vaccines to avoid the safety and effectiveness issues sometimes associated with vaccines based on attenuated viruses and recombinant viral vectors. In addition, methods to manufacture RNA vaccines are amenable to generic platforms and to rapid response, which are critical needs in responding to newly emerging pathogens. Key to the advance of mRNA vaccines has been the ability to diminish the immunogenicity of the nucleic acid and to increase its efficiency (translational capacity). Dr. Zon noted that this was emphasized in a “landmark publication” by Dr. Katalin Kariko et al., in 2008. In earlier work, Dr. Kariko’s group had demonstrated that the use of base-modified triphosphates to enzymatically synthesize in vitro mRNA having modified nucleosides [such as pseudouridine (Ψ), 5-methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), or 2-thiouridine(s2U)] had greatly diminished immunostimulatory properties. They reasoned, Dr. Zon said, that, “if any of the in vitro transcripts containing nucleoside modifications would remain translatable and also avoid immune activation in vivo, such an mRNA could be developed into a new therapeutic tool for both gene replacement and vaccination.” Using the above and other base-modified nucleotide triphosphates—all obtained from TriLink BioTechnologies, Dr. Kariko found that mRNA containing pseudouridine actually had a higher translational capacity than unmodified mRNA. In 2012, Zon said that Kariko et al., demonstrated the real possibility of mRNA therapeutics by showing that non-immunogenic pseudouridine-modified mRNA encoding erythropoietin (EPO) was translated in mice and non-human primates, with results superior to those obtained using EPO mRNA containing uridine. The EPO translated from pseudouridine mRNA was functional and caused significant increase of both reticulocyte counts and hematocrits. In another possibly clinically significant result, Dr. Zon highlights an October 2013 article in Nature Biotechnology that describes highly intriguing research demonstrating greatly improved survival of mice after experimentally induced heart attacks if the mice were given a pulse of RNA therapy. Dr. Zon said that the investigators reported that intra-myocardial injections of vascular endothelial growth factor-A (VEGF-A) mRNA, modified with 5-methylcytidine, pseudouridine, and 5’ cap structure, resulted in expansion and directed differentiation of endogenous heart progenitors in a mouse model of myocardial infarction. They found markedly improved heart function and enhanced long-term survival of recipients. Moreover, “pulse-like” delivery of VEGF-A using modified mRNA was found to be superior to the use of DNA vectors in vivo. Dr. Zon next discussed the recent use of modified mRNA for cellular reprogramming. Dr. Zon notes that the reprogramming of differentiated cells to so-called “induced pluripotent stem cells” (iPSCs) was originally reported by Takahashi and Yamanaka and was achieved by the enforced retroviral expression of four transcription factors (also known as “Yamanaka factors”). Takahashi and Yamanaka shared the 2012 Nobel Prize in Physiology or Medicine for this work, together with John Gurdon, whose much earlier work had provided the intellectual impetus for the seminal creation of iPSCs. Dr. Zon noted that viral integration into the host genome initially presented a significant barrier to the clinical use of these iPSCs and has spurred research into ways to induce pluripotency without risking genetic changes. Along these lines, he said that considerable attention has been focused on a 2010 article by Warren et al., entitled, “Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA.” In this work, complete substitution of either 5-methylcytidine for cytidine or pseudouridine for uridine in protein-encoding transcripts markedly improved protein expression, although the most significant improvement was seen when both modifications were used together. Transfection of modified mRNAs encoding the above mentioned Yamanaka factors led to robust expression and correct localization to the nucleus. The researchers went on to demonstrate that repeated administration of modified mRNA encoding these (and other) factors led to reprogramming various types of differentiated human cells to pluripotency with conversion efficiencies and kinetics substantially superior to established viral protocols. Importantly, Dr. Zon said, this simple, non-mutagenic, and highly controllable technology was shown to be applicable to a range of tissue-engineering tasks, exemplified by mRNA-mediated directed differentiation of mRNA-generated iPSCs to terminally differentiated myogenic (e.g., heart muscle) cells. Dr. Zon noted that a company, Moderna Therapeutics, has been founded based on this technology and the commercial potential is indicated by the fact that AstraZeneca, one of the world’s largest pharmaceutical companies, intends to use Moderna’s technology to develop and commercialize new drugs for cancer and serious cardiovascular, metabolic, and renal diseases in a multi-year arrangement that could bring Moderna more than $420 million. AstraZeneca and Moderna believe that use of what the companies are calling “messenger RNA Therapeutics™” could dramatically reduce the time and expense associated with creating therapeutic proteins using current recombinant technologies. In a related commercial development, Moderna announced on October 2, 2013, that the U.S. Defense Advanced Projects Agency (DARPA) has awarded the company up to $25 million for R&D using Moderna’s modified-mRNA therapeutics platform as a “rapid and reliable way to make antibody-producing drugs to protect against a wide range of now and emerging infectious diseases and engineered biological threats.” Dr. Zon closed by briefly describing a number of other companies, including TriLink BioTechnologies, that have commercial interests in this exciting and potentially vast new space. Dr. Zon is an eminent nucleic acid chemist and Director of Business Development at TriLink BioTechnologies in San Diego, California. The entirety of Dr. Zon’s blog can be viewed at the link below. [Zon blog post]