Speakers

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.

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.

Dr. Richardson works in the standards pipeline development group within Global Biologics at USP, leading efforts to develop standards for emerging technologies such as cell and gene therapy. In previous roles at Advanced BioScience Laboratories and the Foundation Fighting Blindness, he led translational science activities for the development of vaccines and biologics to prevent and treat infectious and retinal diseases. Trained as a virologist, Jim has also held positions responsible for performing viral clearance and adventitious agent testing at Viromed Biosafety as well as AAV vector development and characterization at Genovo/Targeted Genetics. Dr. Richardson earned his PhD in Biomedical Sciences at the Mount Sinai School of Medicine.

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.

James Brown, PhD is Chief of Staff and Vice President, Corporate Development at Aldevron, a biotechnology product and services company. Since joining the company in 2015, his responsibilities include developing new products and services for Aldevron’s lines of business, collaborations and licensing, and pursuing strategic initiatives and partnerships on behalf of the CEO. Prior to his work at Aldevron, Dr. Brown was an early employee of REGENXBIO, helping to establish the company in 2009 and serving until 2015 as Vice President, Technical Operations. At REGENXBIO Dr. Brown was responsible for manufacturing, operations, and quality assurance. Prior to REGENXBIO Dr. Brown held positions of increasing responsibility at MedImmune (AstraZeneca), Meso Scale Discovery, and IGEN International, Inc. Dr. Brown holds a PhD in Chemistry from Stanford University and a BS in Chemistry from Butler University.

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.