Even simple, canonical nucleic acids introduce a variety of challenges for development as therapeutics, including sequence fidelity, susceptibility to damage during production, and stability. The nucleic acid modifications of the epigenome and epitranscriptome have emerged as major biological determinants of nucleic acid function. These DNA and RNA modifications, as well as the introduction of non-biological nucleic acid modifications, add new challenges to bioanalytics during development and clinical trials and to quality control metrics in manufacturing. Over the past three decades, the Dedon Lab has focused on developing quantitative analytical methods for genetic toxicology, epigenetics, and epitranscriptomics. Here I will focus on recent developments in analytical and sequencing methods pertinent to nucleic acid therapeutics. Among analytical methods, mass spectrometry has emerged as the most powerful tool for discovering and quantifying DNA and RNA damage and modifications with high sensitivity. We have also developed DNA and RNA sequencing methods that address damaged products as well as native and synthetic nucleic acid modifications. These methods and technologies can be applied to any step of the drug discovery and development process as well as to manufacturing practices.

Pete Dedon is the Singapore Professor in the Department of Biological Engineering at MIT and the Lead Principal Investigator in the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG). With a research program that applies chemical approaches to nucleic acid biology, his group has developed a variety of analytical and informatic platforms for basic and translational research in epigenetics, epitranscriptomics, and genetic toxicology in infectious disease and cancer. One platform coordinates new sequencing technologies, comparative genomics, and mass spectrometry to discover novel epigenetic marks, such as phosphorothioate, hypoxanthine, and 7-deazaguanine modifications in bacterial and bacteriophage genomes in the human microbiome. In the realm of the epitranscriptome — the dozens of modified ribonucleosides in all forms of RNA — his team has developed and applied systems-level analytics to discover a mechanism of translational regulation of gene expression in bacteria, parasites, mammalian cells, and humans. This response mechanism coordinates stress-specific reprogramming of 40–50 different tRNA modifications with alternative genetic information consisting of biased use of synonymous codons in families of stress response genes. The net result is selective translation of codon-based gene families essential for survival or phenotypic change. Pete and colleagues are leveraging these discoveries to develop new enzymatic tools for biotechnology, new methods for industrial microbiology and protein production, and novel antimicrobial agents and biological therapeutics.

Plasmids and mRNA-based products are increasingly being used as gene therapies in the treatment of cancer, rare diseases, and chronic disorders. These gene therapy products are often delivered as lipid nanoparticle (LNP) complexes. This presentation will cover some of the regulatory aspects of the manufacturing process, in-process testing, and lot release criteria of these products and discuss some of the related regulatory challenges. The presentation will also highlight key considerations and strategies that sponsors may adopt when planning for an Investigational New Drug (IND) application for plasmid and mRNA-based gene therapies.

Dr. Chattopadhyay received her PhD in Cell and Molecular Biology from the University of Maryland in 2015. During her PhD studies, she received an NIH virology training grant to study the mechanistic details of cap-independent translation of positive-sense RNA viruses that are neither 5’-capped nor 3’-polyadenylated. Her research work was also focused on understanding the host’s natural antiviral defense mechanism and its regulation by the viral RNA tertiary structure. Dr. Chattopadhyay received a fellowship from the Oak Ridge Institute for Science and Education to pursue her post-doctoral research work at FDA. She worked on a project that examined the atypical activity properties of factor VIII in hemophilia A gene therapy. Dr. Chattopadhyay has been a full-time gene therapy CMC reviewer at FDA since October 2019.

Messenger (m)RNA has been investigated as a platform technology for multiple therapeutic applications for several years and has now reached the spotlight with two approvals for mRNA-based vaccines against SARS-CoV-2 at the end of 2020. In general, mRNA as a therapeutic modality is applicable whenever expression of a protein — be it as a functional entity or as an antigen — is desired. In my talk I will present the advantages and challenges for manufacturing mRNA for clinical studies. In addition, I will discuss regulatory considerations with respect to the classification of mRNA as a gene therapy by both the FDA and EMA.

Andreas Kuhn has worked in the field of RNA biochemistry and molecular biology for more than thirty years. His work on mRNA-based immunotherapies began in 2007 in the academic group of Ugur Sahin at the University Clinic Mainz, and Andreas joined BioNTech SE shortly after its founding in 2008. In his current role as Senior Vice President RNA Biochemistry & Manufacturing, his main focus is expanding proprietary technologies to increase the efficacy of mRNA-based therapies and to optimize GMP-compatible manufacturing processes for RNA. He has co-authored numerous publications and patents ranging from basic research on RNA to its application as a diagnostic and therapeutic agent.

• Topology Band Identification
• Inverted Terminal Repeats
• Nucleic Acid Cross Contamination
• Site Specific Damage

Lawrence (Larry) Casper Thompson, PhD is a Senior Principal Scientist & Group Leader in Analytical R&D within BioTherapeutic Pharmaceutical Sciences at Pfizer. He has been with Pfizer for over seven years and is currently CMC analytical lead for Pfizer’s adenoviral & plasmid DNA-based immunotherapeutic and nucleic acid starting material pipeline (used for both rAAV gene therapy and mRNA vaccine manufacturing). Prior to joining Pfizer, he spent three years in small biotech at two different companies as lead scientist in the development of serum-based cancer diagnostics. He received his PhD in Biochemistry from Vanderbilt University and did his postdoctoral work at the University of Tennessee. His work has generated a number of peer reviewed publications and presentations at scientific conferences, as well as internally within Pfizer.

Demand for DNA has risen dramatically in recent years, driven primarily by demand for viral vector production and cell therapy. Additionally, the rapid development of nucleic acid (mRNA, DNA) vaccines in response to the COVID-19 pandemic has further driven demand for DNA, exacerbating a significant bottleneck in industry capacity.

Touchlight has developed dbDNA, an enzymatically amplified DNA vector with demonstrated utility in production of viral vectors, mRNA, cell therapy, and as a DNA vaccine. dbDNA technology can be manufactured rapidly at industrial scale, and can help eliminate the existing bottlenecks in DNA supply, further enabling the genetic medicine revolution.

Antonio Marques, PhD is the Chief Operating Officer for Touchlight DNA Services, leading the operations of the CDMO division of Touchlight. Prior to Touchlight, he had senior management positions in medical devices and in vitro diagnostics, followed by a move to the service provision industry as Managing Director for a large molecule bioanalysis CRO. Antonio received a PhD in biochemistry and molecular biology from the University of Cologne, Germany.

Minicircle DNA gains increasing importance for clinical research applications in gene therapy and genetic vaccination. AAV vectors and CAR T-cells are two very promising tools since both have already successfully entered the clinic. For direct gene transfer into humans, GMP-grade DNA is mandatory. Whereas in many cases high quality (HQ) grade DNA is accepted as a starting material, in GMP production of mRNA or viral vectors for example. If the therapeutically active substance is a genetically modified cell, the situation is currently actively discussed. Here the DNA may either serve as a starting material or be part of the drug substance.

Compared to the corresponding plasmids, minicircles provide a striking benefit, especially for production of ATMP candidates such as AAV vectors, CAR T-cells, and hematopoietic stem cells. In the case of AAV vector production, minicircles resulted in a higher purity of the packaged viral particles. For CAR T-cell production based on the Sleeping Beauty system, minicircles resulted in an improved transposition rate and less genotoxicity.

Consequently, in close collaboration with the leading experts in the fields of AAV and CAR T-cell-based therapies, we have developed a process for the production of HQ grade minicircle DNA. In a dedicated facility, starting from a characterized research cell bank (RCB), the manufacturing process passes through different well-documented production steps. The HQ fermentation facility is completely separated from purification (chromatography). The proprietary purification procedure results in pure, supercoiled (ccc) minicircle monomers, meeting the regulatory requirements of a defined, homogenous product, proven by a number of quality controls on the cell bank as well as on the minicircle DNA product.

Dr. Ram Shankar completed his PhD in 2014 at the Chair of Fermentation Engineering, Bielefeld University in Germany. His doctoral work focused on the extracellular expression of a recombinant enzyme from Escherichia coli in continuous cultivation mode while employing antibiotic-free plasmid selection mechanisms. Thereafter, he started as a postdoc at PlasmidFactory GmbH & Co. KG moving on to become Manager R&D in 2016. At PlasmidFactory, his responsibilities include managing a wide array of research projects aimed at improving plasmid production and purification processes. These include strain engineering, recombinant enzyme production, development of new upstream and downstream processes, optimization of purification processes, and development of analytical methods for quality control.

Established processes for pDNA manufacturing include purification steps designed to enrich the supercoiled (sc) isoform. Homogeneous supercoiled isoform improves cell transfection efficiency in fermentation processes and is favoured in cell culture production systems. In vitro transcription (IVT), the enzymatic process used for production of mRNA vaccines, differentiates itself from biological fermentation processes by the need for linearised plasmid DNA. The linear isoform is produced with restriction enzymes from open circular and supercoiled plasmid DNA. Employing a traditional pDNA manufacturing process, which removes linear and open-circular isoforms, will therefore reduce the production yield. When plasmid DNA and mRNA are treated as a single production process, the purification steps can be optimised, yields improved, and the overall production cost reduced.

A new purification approach starting from E. coli through to mRNA production is presented here. This new approach integrates a pDNA linearisation step before polishing (removal of linear and open circular isoforms) of plasmid DNA. The polishing step, placed after enzymatic linearisation, purifies linear pDNA from enzyme and other unwanted process impurities. The linearised plasmid DNA is then used in IVT for production of mRNA. Fast in-process analytics allows for transcription number well above 100 in a reproducible manner.

This approach further introduces mRNA purification tools for improved contaminant removal. The altered sequence of purification and linearisation reduces the overall number of purification steps required and improves recoveries, while the complete process results in extra-low protein impurity and very efficient dsRNA removal.

Pete Gagnon is a widely known and respected authority in the field of downstream processing. He holds more than 100 patents in more than a dozen countries, covering various aspects of purifying antibodies, other recombinant proteins, virus particles, and exosomes. He is the author of more than 100 articles, book chapters, and books; an editorial advisor for several journals and conferences; and a frequent conference presenter. He has held high level positions in several companies and is currently the Chief Scientific Officer of BIA Separations.

Plasmid DNA is a material that is increasingly being used to manufacture advanced therapy products. United States Pharmacopeia (USP) has been engaging with stakeholders to explore the issues associated with using plasmid DNA in manufacturing of cell and gene therapies. To this end, we have formed an expert panel that is charged with developing a new informational chapter to be included in the USP-National Formulary (NF). It is expected that this chapter will cover regulatory considerations, sourcing/vendor qualification, and characterization of plasmid DNA for use in manufacturing. This presentation will cover the framework for the chapter, outlining the issues that developers have encountered while using plasmid DNA as well as possible solutions. We will provide an update on the progress to date and identify areas where additional feedback is needed.

Nate Spangler leads product and service initiatives for the production of enzymes and mRNA for cancer immunotherapy, gene therapy, and cell therapy. Nate is a graduate of the University of Wisconsin-Madison with a PhD in Biochemistry.