Bio

David Brown, PhD, has research interests focused on using synthetic biology tools to advance virology research and vaccine development. He began his graduate work by seeking a more rapid method of producing influenza vaccine seeds from oligonucleotide assembly in collaboration with Novartis Vaccines and Diagnostics and the JCVI. As part of his PhD dissertation, he developed a method of delivering large synthetic DNA molecules, and synthetic proteins, to mammalian tissue cultured cells via yeast fusion, which was capable of being used as a reverse genetics system for HSV-1. He is currently engaged in work generating human artificial chromosomes, developing assays for viral pathogenesis, and characterizing contemporary EV-D68 strains.

Dr. Brown received a BA in Biochemistry and Molecular Biology from Hamilton College, NY. He received a PhD in Biology from the University of Maryland at College Park in 2017, while a research fellow at the JCVI.

Research Priorities

Delivery of DNA from yeast to tissue cultured cell lines. Delivery of transgenic DNA into mammalian cells is critical to realizing the potential of synthetic biology. Better DNA delivery technology will have applications in advancing gene therapy, construction of entire chromosomes, and production of new vaccines and therapeutics in cultured mammalian cells. New synthetic biology techniques such as rapid, inexpensive DNA synthesis have opened the door to engineering biology. However, now the delivery of these synthetic DNA constructs to the nucleus of a living cell is the limiting step in the development of these applications.

  • We developed a method of transferring large DNA molecules cloned in Saccharomyces cerevisiae into cultured cells through polyethylene glycol mediated fusion of the yeast and cultured cells.
  • Increased the delivery efficiency of large YCps (up to 1.1 Mb) using a design of experiments approach ten-fold to 1/1000
  • This method was adapted to deliver a 152-kb herpes simplex virus genome cloned in yeast into mammalian cells to produce infectious virus.

Rapid Tests for Virus Genes that Suppress the Host Antiviral Defenses. Identifying viral proteins that disrupt the infected host’s antiviral response systems has been a major challenge. It is difficult to identify such “host defense inhibitor” genes solely from genomic sequence. Viral genes and proteins vary greatly in sequence across families, can be multifunctional, and may act both as essential elements for viral replication and inhibitors of host. These viral proteins play a major role in pathogenicity – the more the immune system is disabled, the longer it takes to fight off an infection, and the greater the chance of severe disease or fatality. We are developing assays to screen for these host defense inhibitors.

  • Using yeast delivery, we have developed a prototype assay for detecting a host defense inhibitor on non-permissive cells.
  • We will be constructing a library of approximately 1000 genes encoding all the genes from a set of human viruses across all known families that infect humans and test each gene for its capacity to permit replication of at least one DNA and one RNA virus on non-permissive cells.

Genetic Determinants of Neurovirulence in Recent Enterovirus D68 Outbreak. Historically, enterovirus D68 (EV-D68) has only been associated with respiratory illnesses. However, in the summer of 2014 and 2016 EV-D68 outbreaks coincided with a spike in polio-like Acute Flaccid Myelitis/Paralysis (AFM) cases. Statistical analysis suggested that the number of AFM cases was significantly higher during the EV-D68 outbreak than in historical controls. We have theorized that members of the B1 subclade are responsible for the observed neurotropism and are seeking to characterize the genetic determinants of the neurotropic phenotype.

  • We have developed a neuroblastoma-derived neuronal cell line as a cell culture mode that show differential replication of historic and contemporary neurotropic EV-D68 strains
  • We have developed a reverse genetics method for EV-D68 production using RNA transfection
  • Synthetic swap mutants using contemporary EV-D68 stains are being built and characterized to identify which genetic determinants are responsible for the observed neurotropic phenotype