Cambridge Healthtech Institute’s Ninth Annual
Engineering Genes and Hosts
Strategies in Systems and Synthetic Biology
January 9-10, 2017 | Hilton San Diego Bayfront | San Diego, CA
Engineering therapeutic protein expression platforms is not for the faint of heart. Many variables must be considered during the engineering process, including verification and sequence analysis of the gene or protein of interest, codon optimization,
vector construction and clone/host selection. When challenges arise, protein expression engineers must design new cloning schemes by altering the DNA or amino acid sequence, moving a gene from one vector to another, transfecting the vector to
an alternative host, re-selecting the clone, re-characterizing the expressed protein or any of the above – a laborious, time-consuming and expensive process.
Cambridge Healthtech Institute’s Ninth Annual Engineering Genes and Hosts conference continues the tradition of applying effective engineering strategies for protein expression and production research leading to functional biotherapeutic products.
Learn from seasoned, savvy researchers as they share their real-world experiences, applications and results.
MONDAY, JANUARY 9
am Conference Registration and Morning Coffee
9:00 Welcome by Conference Organizer
Mary Ann Brown, Executive Director, Conferences & Team Lead, PepTalk, Cambridge Healthtech Institute
9:05 Chairperson’s Opening Remarks
Henry C. Chiou, Ph.D., Associate Director, Cell Biology, Life Science Solutions, Thermo Fisher Scientific
9:10 Systems Engineering as a Strategy for Developing New Therapeutics against Infectious Diseases and Cancers
Nitin S. Baliga, MSc, Ph.D., Professor, Senior Vice President & Director, Institute for Systems Biology
Disease is manifestation of dysfunction in complex cellular and molecular networks. A systems approach is necessary to elucidate causal and mechanistic underpinnings of dysfunctional biological networks. I discuss how we use a systems approach
to elucidate dysfunctional networks in human and microbial systems, and how that understanding is being used to discover new personalized therapies in brain cancer and fight drug tolerance in TB.
9:50 High-Throughput Multiplexed Genome Editing: Forgecraft
Eileen Spindler, Ph.D., Director, R&D Innovation, MUSE Biotechnology, Inc.
Advances in DNA synthesis and sequencing have motivated increasing efforts to program cells on laboratory timescales. While CRISPR-based methods for genome editing are extremely efficient, strategies for parallel editing throughout a genome
have been limited. Here we describe CRISPR EnAbled Trackable genome Engineering (CREATE), a strategy that couples the high efficiency of CRISPR editing with massively parallel oligomer synthesis to perform precise, trackable editing on
a genome-wide scale.
10:20 Coffee Break
10:45 Cell-Free Protein Synthesis: A Versatile Enabling Technology for Synthetic Biology
Rui Gan, Ph.D., Research Associate, Michael C. Jewett Laboratory, Chemical and Biological Engineering, Northwestern University
Cell-free protein synthesis (CFPS) has been widely applied to express various proteins due to its high yield and easy manipulation. In synthetic biology, this technology is found to be extremely powerful in directed evolution, metabolism network
analysis, genetic circuit construction, and assembly of complex macromolecules. Combined with a microfluidics system, CFPS can be encapsulated into emulsion droplets to perform automatic analysis and evolution of enzymes.
11:15 Reinforcing Synthetic Biology against Evolutionary Failure Modes
Jeffrey E. Barrick, Ph.D., Assistant Professor, Molecular Biosciences, The University of Texas
Unwanted evolution makes genetic engineering less predictable and reliable. In particular, takeover of cultures by “cheater” cells with mutations that disrupt an engineered function can be a major problem in scaling up processes.
I discuss high-throughput methods for profiling these evolutionary failure modes, how computational design can avoid inherently unstable DNA sequences, and the results of using directed evolution to isolate host cells that have lower-than-natural
11:45 A Semi-Synthetic Organism with an Expanded Genetic Alphabet
Floyd Romesberg, Ph.D., Professor, Chemistry, The Scripps Research Institute
We have developed an unnatural base pair and recently reported its use in E. coli to create the first semi-synthetic organism that stores increased genetic information. Recent progress will be discussed
that has resulted in a healthy semi-synthetic organism capable of the indefinite storage of multiple unnatural base pair. Progress toward the retrieval of that information via transcription and translation will also be discussed.
12:15 Vitamin B5 Transport as a Metabolic Selection for Highly Efficient Recombinant Protein Expression by Mammalian Cells
Lucille Pourcel, Ph.D., Postdoc Laboratory, Molecular Biology, University of Lausanne
We designed an improved selection method based on the co-expression of a vitamin B5 transporter, relying on mammalian cell dependence on this vitamin for energy production. This method yields polyclonal cell populations producing recombinant
proteins at homogeneous and high level, using the selective advantage of improved cell metabolism, growth and viability. This selection is also efficient to select variant cells synthesizing difficult-to-express chimerical proteins
at elevated levels.
12:45 Session Break
1:00 Luncheon Presentation: Engineering Biology on DNA, Protein and Genome Level
James Love, Ph.D., Director, Expression Technologies, Protein Expression, ATUM (formerly DNA2.0)
Modern machine learning combined with efficient gene synthesis allow for unprecedented ability to engineer biological systems at the protein, gene, vector, and genome levels with absolute precision. Transient and stable CHO/HEK
protein expression yield is controlled over orders of magnitude by the systematic incorporation of validated sequence elements. The presentation will review how DNA2.0 engineering tools are leveraged for the rapid and efficient
production and optimization of biotherapeutics.
2:00 Chairperson’s Remarks
Mark Welch, Ph.D., Vice President, Research and Development, ATUM (formerly DNA2.0)
2:05 Targeted Isolation and Cloning of 100-kb Microbial Genomic Sequences by Cas9-Assisted Targeting of Chromosome Segments
Ting Zhu, Ph.D., Investigator & Associate Professor, School of Life Sciences, Tsinghua University
The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. We have developed a technique (CATCH) that allows
the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. This technique can be an effective molecular tool for the targeted cloning of large gene clusters.
2:35 CRISPR/Cas9-Mediated Multiplex Genome Editing for Efficient CHO Cell Engineering
Byung-Kwan Cho, Ph.D., Associate Professor, Biological Sciences, Korea Advanced
Institute of Science and Technology (KAIST)
Efficient and rational CHO cell engineering methods have been in high demand to improve quality and productivity. Here, we provide a novel genome-engineering platform for increasing desirable phenotypes of CHO cells based upon
the integrative protocol of high-throughput RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR-associated protein 9) nuclease-based genome editing.
3:05 Design CRISPR/Cas9 in a Unified Software Platform: Transforming Genome Engineering with Benchling
Sajith Wickramasekara, Founder and CEO, Benchling
CRISPR/Cas9 has sparked a revolution in genome editing, but existing solutions force users to engage with disparate pieces of software. In this talk we will describe how we worked with scientists to empower them to design, register
and optimize their genome engineering process on a single collaborative research platform.
3:20 Refreshment Break in the Exhibit Hall with Poster Viewing
4:00 Versatile Genome Engineering via CRISPR-Cas Systems
Ana Moreno, Research Scientist, Bioengineering, University of California, San Diego
Technologies to directly and precisely perturb genomic elements and combinations will be a critical toolset towards obtaining the complete functional annotation of genomic elements and genetic variants at the cellular and whole
organism levels, and also to program the genome for medicinal or technological purposes. This talk provides an overview of the rapidly developing CRISPR-Cas genome engineering toolset, with a specific focus on therapeutic applications.
4:30 A High-Throughput Method for Parsing Complex DNA Libraries and Engineering Yeast
Robert St. Onge, Ph.D., Senior Research Scientist, Stanford Genome Technology Center,
We have developed an inexpensive, high-throughput method for parsing and sequence-verification of array-synthesized oligonucleotide libraries, and are using it to produce DNA probes for molecular detection, guide RNAs for CRISPR/Cas9-based
genome editing and programmable transcription, and building blocks for gene synthesis. The method allowed rapid creation of ~9,000 individually accessible CRISPR interference strains for the essential yeast genome. Applications
for host and genome engineering will be presented.
5:00 CRISPR-Cas9 Tools for the Baculovirus-Insect Cell System
Hideaki Mabashi-Asazuma, Ph.D., Research Scientist, Molecular
Biology, University of Wyoming
This study examines the utility of previously and newly identified insect U6 promoters for CRISPR-Cas editing of insect cell lines used as hosts for baculovirus vectors. We discovered surprisingly tight cross-species restrictions,
which dictated the utility of different insect U6 promoters for CRISPR-Cas editing in different insect species. Ultimately, we identified efficacious U6 promoters for each host and a novel lepidopteran insect-specific sequence
element required for optimal U6 promoter function.
6:20-7:30 Welcome Reception in the Exhibit Hall with Poster Viewing
7:30 Close of Day
TUESDAY, JANUARY 10
am Conference Registration and Morning Coffee
8:30 Chairperson’s Remarks
James Brady, Ph.D., Director, Technical Applications, MaxCyte, Inc.
8:35 Improvements to the Baculovirus-Based Insect Cell Expression System to Enhance Protein Yield and Quality
Dominic Esposito, Ph.D., Director, Protein Expression Laboratory, Frederick National
Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Insect cell systems have become more prevalent for the production of pharmaceutically relevant protein targets in the last decade. In our laboratory, this system has been vital for production of post-translationally modified RAS
proteins essential for cancer drug discovery. In the process of utilizing this system, we have developed a number of process and technology improvements which permit increased protein yield, protein quality, and virus stability.
We discuss in detail the enhancements to the system and how they can be applied to high-level production of other clinically relevant proteins, and examine ways in which synthetic biology and genome engineering can further
enhance the utility of this system.
9:05 Engineering Recombinant mRNAs for Increased Protein Expression, Better Secretion, and Improved Cell Physiology
Stephen Chappell, Ph.D., Scientific Director, Promosome, LLC
Expression of a recombinant mRNA, e.g., in cell culture for bioproduction or in vivo for nucleic acid therapeutics and vaccines, is often limited by specific features of the mRNA that can decrease
translation efficiency and negatively affect cell physiology. We have developed a method termed RESCUE™ to eliminate these negative features. RESCUE™-based modifications increase protein expression and secretion
while minimizing conformational changes that can alter biological activity or cause immunogenicity.
9:35 Selected Poster Presentation: Intracellular Secretion Pathway Analysis for Constructing Highly Producible Engineered CHO Cells
Kohei Kaneyoshi, Research Scientist, Graduate School of Engineering, Osaka University
Though the CHO cell is widely used for the production of therapeutic antibodies, the secretion of recombinant antibodies is not fully investigated. We analyzed the secretion of IgG in engineered CHO cells by chase assay. It revealed
the secretion reached a plateau, even though some IgG still remained in the cells. The remaining IgG co-localized mainly with endoplasmic reticulum. Furthermore, a part of them had immature forms, which should be improved for
constructing high producers.
9:50 Coffee Break in the Exhibit Hall with Poster Viewing
11:15 Selected Poster Presentation: Towards Better Cell Factories for Bioproduction - HEK293
Magdalena Malm, Ph.D., Lab Manager, Wallenberg Centre for Protein Research, KTH Royal Institute of Technology
With the rapid increase in biopharmaceuticals entering the market, there is a great need for bioproduction platforms enabling both high titers and high quality. The Wallenberg Centre for Protein Research (WCPR) is a multidisciplinary
center with the aim to develop new biopharmaceuticals as well as improved technologies for their production. In this center, we have analyzed the expression and secretion of various difficult to express proteins in the human
cell line HEK293 in order to evaluate this cell line as an alternative to the conventional CHO cells. Furthermore, we have generated stable HEK293 clones expressing a model protein (EPO) to use as tools to characterize HEK293
as a bioproduction strain as well as understand differences between high and low producers.
11:30 Engineering Mammalian Cells for Desired Bioprocess and Protein Quality Attributes
Nathan E. Lewis, Ph.D., Assistant Professor, Pediatrics, University of California, San Diego
In mammalian bioprocessing, product quality can vary widely, given the diverse nature of cells, even in seemingly clonal populations. Genomic and computational tools now allow us to understand mammalian host cells, and powerful
genome editing systems allow us to engineer desired traits. I discuss efforts in which we have used systems biology techniques to design host cells to control glycosylation, protein yields and improved bioprocess traits.
12:00 pm Vmax™ – a Next-generation Microbial Workhorse for the Biotech Industry
Matthew Weinstock, Scientist II, Research Systems, Synthetic Genomics
This presentation will focus on Vmax™, a rapidly growing and highly productive prokaryotic host that promises to accelerate biotech R&D efforts on multiple fronts. We will describe the development of
the platform, the advantages of using it in molecular cloning and protein expression applications, and ongoing large-scale genome engineering efforts to further enhance performance.
12:30 Session Break
12:45 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own
1:15 Close of Conference